EP3110441A1 - Nouveau polysaccharide et utilisations associées - Google Patents

Nouveau polysaccharide et utilisations associées

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Publication number
EP3110441A1
EP3110441A1 EP15706459.3A EP15706459A EP3110441A1 EP 3110441 A1 EP3110441 A1 EP 3110441A1 EP 15706459 A EP15706459 A EP 15706459A EP 3110441 A1 EP3110441 A1 EP 3110441A1
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EP
European Patent Office
Prior art keywords
coli
antigen
specific embodiment
host cell
carrier protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP15706459.3A
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German (de)
English (en)
Other versions
EP3110441B1 (fr
Inventor
Michael T. KOWARIK
Michael L. WETTER
Stefan J. KEMMLER
Micha A. HÄUPTLE
Veronica Gambillara
Manuela Mally
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GlaxoSmithKline Biologicals SA
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Glycovaxyn AG
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Publication of EP3110441A1 publication Critical patent/EP3110441A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0066—Isolation or extraction of proteoglycans from organs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • E. coli antigen 025B Disclosed herein are the structure of the E. coli antigen 025B, as well as uses of 025B, methods of making of 025B, and bioconjugates comprising 025B. Applicants have identified the E. coli gene cluster responsible for production of 025B and have fully characterized the structure of the 025B antigen. Accordingly, provided herein are nucleic acids capable of producing 025B in host cells. Also provided herein are host cells, e.g., recombinantly engineered host cells, comprising nucleic acids capable of 025B production.
  • Such host cells can be used to generate bioconjugates comprising 025B linked to a carrier protein, which can be used in, e.g., the formulation of therapeutics (e.g., vaccines).
  • the 025B antigen described herein also is useful in the generation of antibodies, which can be used, e.g., in therapeutic methods such as passive immunization of subjects.
  • compositions comprising 025B, alone or in combination with other E. coli antigens (e.g., 01, 02, and 06 and subserotypes thereof), for use in therapeutic methods, e.g., vaccination of hosts against infection with E. coli (e.g., extra-intestinal pathogenic, such as uropathogenic, E. coli).
  • Extra-intestinal pathogenic E. coli causes a wide variety of infections that are responsible for significant morbidity, mortality, and costs annually. Urinary tract infections are among the most frequent conditions caused by ExPEC in human beings.
  • a prokaryotic host cell comprising nucleic acids encoding enzymes (e.g., glycosyltransferases) capable of producing the novel polysaccharide disclosed herein, E. coli 025B.
  • host cells comprising nucleic acids encoding enzymes (e.g., glycosyltransferases) capable of producing other E. coli antigens, e.g., 025 A, 01, 02, and 06, and subserotypes thereof.
  • the host cells provided herein may naturally express nucleic acids specific for production of an O antigen of interest, or the host cells may be made to express such nucleic acids, i.e., in certain embodiments said nucleic acids are heterologous to the host cells.
  • the host cells provided herein comprise nucleic acids encoding additional enzymes active in the N-glycosylation of proteins, e.g., the host cell provided herein can further comprise a nucleic acid encoding an oligosaccharyl transferase or nucleic acids encoding other glycosyltransferases.
  • the host cells provided herein comprise a nucleic acid encoding a carrier protein, e.g., a protein to which oligosaccharides and/or polysaccharides can be attached to form a bioconjugate.
  • a carrier protein e.g., a protein to which oligosaccharides and/or polysaccharides can be attached to form a bioconjugate.
  • the host cell is E. coli. See Section 5.3.
  • a prokaryotic host cell comprising an E. coli r 3 ⁇ 4(upecl38) gene cluster (SEQ ID NO: 12), or a gene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to an E. coli r 3 ⁇ 4(upecl38) gene cluster (SEQ ID NO: 12).
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell comprising an E. coli r 3 ⁇ 4(upecl63) gene cluster, or a gene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to an E. coli r 3 ⁇ 4(upecl63) gene cluster.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • provided herein is a prokaryotic host cell comprising an E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell recombinantly engineered to comprise e.g., by introduction of one or more vectors/plasmids into the host cell) one, two, three, four, or more of the following genes (or a nucleic acid that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to one of the following genes): rmlB (SEQ ID NO: l), rmlD (SEQ ID NO:2), rmlA (SEQ ID NO:3), rmlC (SEQ ID NO:4), wzx (SEQ ID NO:5), wekA (SEQ ID NO:6), wekB (SEQ ID NO:7), wzy (SEQ ID NO:8), wbbJ (SEQ ID NO:9), wbbK (SEQ ID NO: 10), and/or wbbL
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase (e.g., a heterologous oligosaccharyltransferase).
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • the prokaryotic host cells provided herein comprise a deletion or functional inactivation of one or more genes. See Section 5.3.1.
  • one or more of the waaL gene, gtrA gene, gtrB gene, gtrS gene, or the rfb gene cluster is deleted or functionally inactivated from the genome of a prokaryotic host cell provided herein.
  • the carrier proteins expressed by the prokaryotic host cells provided herein can be selected from any carrier proteins known to those of skill in the art, e.g., detoxified Exotoxin A of P. aeruginosa (EPA; see, e.g., Ihssen, et al, (2010) Microbial cell factories 9, 61), CRM 197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, E. coli FimH, E. coli FimHC, E. coli heat labile enterotoxin, detoxified variants of E.
  • detoxified Exotoxin A of P. aeruginosa EPA; see, e.g., Ihssen, et al, (2010) Microbial cell factories 9, 61
  • CRM 197 maltose binding protein (MBP), Diphtheria toxoid,
  • the carrier protein expressed by a prokaryotic host cell provided herein is detoxified Pseudomonas exotoxin (EPA).
  • the carrier protein of a host cell provided herein comprises a signal sequence for targeting the carrier protein into the periplasmic space of the host cell.
  • the signal sequence is from E. coli DsbA, E. coli outer membrane porin A (OmpA), E. coli maltose binding protein (MalE), Erwinia carotovorans pectate lyase (PelB), Flgl, NikA, or Bacillus sp. endoxylanase (XynA), heat labile E. coli enterotoxin LTIIb, Bacillus endoxylanase XynA, or E. coli flagellin (Flgl).
  • the nucleic acid sequence encoding the carrier protein expressed by the host cells provided herein has been engineered (e.g., via recombinant techniques) to encode one or more of the consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); and/or the consensus sequence Asp(Glu)-X-Asn-Z- Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the carrier proteins expressed by the host cells provided herein comprise two, three, four, five or more of said consensus sequences. See Section 5.3.2.
  • an N-glycosylated carrier protein also referred to herein as a bioconjugate
  • a carrier protein e.g., EPA
  • E. coli O antigen e.g., E. coli 025B
  • bioconjugates produced by the host cells provided herein.
  • a bioconjugate comprising a carrier protein (e.g., EPA) N-linked to E. coli 025B. See Section 5.4.
  • EPA carrier protein
  • a bioconjugate comprising a carrier protein (e.g., EPA) linked to a compound of Formula 025B, presented below:
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • the carrier protein is N- linked to the O antigen of Formula 025B.
  • bioconjugate comprising a carrier protein (e.g., EPA) linked to a compound of Formula 025B', presented below:
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • the carrier protein is N- linked to the O antigen of Formula 025B'.
  • an isolated O antigen from an ExPEC E. coli strain wherein said strain produces 025B.
  • an isolated O antigen from E. coli strain upecl38 In another specific embodiment, provided herein is an isolated O antigen from E. coli strain upecl63. In another specific embodiment, provided herein is an isolated O antigen from E. coli strain upecl77. See Section 5.2.
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • n of at least 80% of the macro molecules in the population is between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • n of at least 80% of the macro molecules in the population is between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • compositions e.g., pharmaceutical compositions, comprising the bioconjugates provided herein and/or the macro molecules (or populations thereof) provided herein. See Section 5.6.
  • composition e.g., a pharmaceutical composition, comprising a macromolecule comprising a structure of Formula 025B:
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • composition e.g., a
  • composition comprising a macromolecule comprising a structure of Formula
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • a composition e.g., a pharmaceutical composition, comprising a bioconjugate described herein, wherein said bioconjugate comprises a carrier protein (e.g., EPA) linked to a compound of Formula 025B, presented below:
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • the carrier protein is N- linked to the O antigen of Formula 025B'.
  • composition e.g., a pharmaceutical composition, comprising a bioconjugate described herein, wherein said bioconjugate comprises a carrier protein (e.g., EPA) linked to a compound of Formula 025B', presented below:
  • a carrier protein e.g., EPA
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • the carrier protein is N- linked to the O antigen of Formula 025B'.
  • the pharmaceutical compositions provided herein comprise one or more additional E. coli O antigens, wherein said antigens are not 025B (e.g., the formula 025B or the formula 025B'), e.g., O antigens from E. coli (e.g., ExPEC) other than those from an E. coli 025B serotype, and/or one or more bioconjugates comprising a carrier protein linked to an E. coli O antigen, wherein said antigen is not 025B (e.g., the formula 025B or the formula 025B').
  • 025B e.g., the formula 025B or the formula 025B'
  • O antigens from E. coli e.g., ExPEC
  • bioconjugates comprising a carrier protein linked to an E. coli O antigen, wherein said antigen is not 025B (e.g., the formula 025B or the formula 025B').
  • compositions may comprise one or more additional macro molecules comprising an ExPEC O antigen and/or one or more additional bioconjugates, e.g., an 01 A, 02, and/or 06 macromolecule and/or an 01 A, 02, and/or 06 bioconjugate.
  • additional macro molecules comprising an ExPEC O antigen and/or one or more additional bioconjugates, e.g., an 01 A, 02, and/or 06 macromolecule and/or an 01 A, 02, and/or 06 bioconjugate.
  • composition e.g., a pharmaceutical composition, comprising one or more additional macromolecules comprising an ExPEC O antigen and/or one or more additional bioconjugates, in addition to an 025B macromolecule (e.g., a macromolecule comprising the formula 025B or the formula 025B') and/or an 025B bioconjugate (e.g., a bioconjugate comprising a carrier protein linked to the formula 025B or the formula 025B'), wherein said additional macromolecules comprise a structure selected from the group consisting of:
  • compositions e.g., a pharmaceutical composition, comprising one, two, three, four, five, six, or seven macromolecules or bioconjugates comprising said macromolecules, in addition to an 025B macromolecule (e.g., a macromolecule comprising the formula 025B or the formula 025B') and/or an 025B bioconjugate (e.g., a bioconjugate comprising a carrier protein linked to the formula 025B or the formula 025B'), wherein said additional macromolecules comprise a structure selected from the group consisting of:
  • a composition e.g., a pharmaceutical composition, comprising (i) an 025 (e.g., 025A or 025B) macromolecule, or a bioconjugate comprising 025 (e.g., 025A or 025B) and (ii) an 01 macromolecule or a bioconjugate comprising 01.
  • said 025 macromolecule is an 025B macromolecule.
  • said 01 macromolecule is 01 A.
  • said 01 macromolecule is OIB.
  • said 01 macromolecule is OIC.
  • composition e.g., a pharmaceutical composition, comprising (i) an 025 (e.g., 025A or 025B) macromolecule, or a bioconjugate comprising 025 (e.g., 025A or 025B) and (ii) an 02 macromolecule or a bioconjugate comprising 02.
  • said 025 macromolecule is an 025B macromolecule.
  • composition e.g., a pharmaceutical composition, comprising (i) an 025 (e.g., 025A or 025B) macromolecule, or a bioconjugate comprising 025 (e.g., 025A or 025B) and (ii) an 06 macromolecule (e.g., an 06 macromolecule comprising a branching Glc monosaccharide (06Glc) or a branching GlcNAc monosaccharide(06GlcNAc)) or a bioconjugate comprising 06.
  • said 025 macromolecule is an 025B macromolecule.
  • said 06 macromolecule is an 06 macromolecule comprising a branching Glc monosaccharide (06Glc).
  • composition e.g., a pharmaceutical composition, comprising at least two of the following: (i) an 025 (e.g., 025A or 025B) macromolecule or a bioconjugate comprising 025 (e.g., 025 A or 025B); (ii) an 01 macromolecule or a bioconjugate comprising 01; (iii) an 02 or a bioconjugate comprising 02; and/or (iv) an 06 macromolecule (e.g., a 06 macromolecule comprising a branching Glc monosaccharide or a branching GlcNAc monosaccharide) or a bioconjugate comprising 06.
  • said 025 macromolecule is an 025B macromolecule.
  • said 01 macromolecule is 01 A.
  • said 01 macromolecule is OIB.
  • said 01 macromolecule is OIC.
  • said 06 macromolecule is an 06 macromolecule comprising a branching Glc monosaccharide (also referred to herein as 06Glc).
  • composition e.g., a pharmaceutical composition, comprising an 025B macromolecule, an 01 A macromolecule, an 02 macromolecule, and an 06 macromolecule comprising a branching Glc monosaccharide.
  • said macromolecules are conjugated to carrier proteins.
  • a composition e.g., an immunogenic composition described herein. See Section 5.7.
  • a composition e.g., an immunogenic composition
  • a composition e.g., an immunogenic composition described herein. See Section 5.7.
  • opsonophagocytic antibodies against ExPEC in a subject, e.g., a human subject, comprising administering to the subject a pharmaceutically effective amount of a composition (e.g., an immunogenic composition) described herein. See Section 5.7.
  • a composition e.g., an immunogenic composition
  • OPS O polysaccharide; the O antigen of Gram-negative bacteria. OPS also are referred to herein as O antigen.
  • rfb cluster a gene cluster (e.g., an E. coli gene cluster) that encodes enzymatic machinery capable of synthesis of an O antigen backbone structure.
  • the term rfb cluster may apply to any O antigen biosynthetic cluster, including those from bacteria that do not belong to genus Escherichia.
  • waaL the O antigen ligase gene encoding a membrane bound enzyme with an active site located in the periplasm.
  • the encoded enzyme transfers undecaprenylphosphate (UPP)- bound O antigen to the lipid A core, forming lipopolysaccharide.
  • UFP undecaprenylphosphate
  • wecA the first gene encoded in the wee cluster.
  • the encoded protein catalyzes the transfer of a GlcNAc-phosphate from UDP-GlcNAc to UPP to form UPP-bound GlcNAc.
  • ECA enterobacterial common antigen
  • RU repeat unit. As used herein, the RU is set equal to the Biological repeat unit, BRU.
  • BRU describes the RU of an O antigen as it is synthesized in vivo.
  • UPP undecaprenylpyrophosphate.
  • LLO lipid linked oligosaccharide.
  • MS mass spectroscopy
  • 025B refers to the 025B antigen from E. coli identified herein (a subserotype of E. coli serotype 025). Reference to 025B herein encompasses the formula 025B and the formula 025B', both identified above.
  • 025A refers to the 025A antigen of E. coli (a subserotype of E. coli serotype 025). Reference to 025A herein encompasses the formula 025A and the formula 025 A', both identified above.
  • 01 A refers to the OIA antigen of E. coli (a subserotype of E. coli serotype 01). Reference to OIA herein encompasses the formula OIA and the formula OIA', both identified above.
  • OIB refers to the OIB antigen of E. coli (a subserotype of E. coli serotype 01). Reference to OIB herein encompasses the formula OIB and the formula OIB', both identified above.
  • OIC refers to the OIC antigen of E. coli (a subserotype of E. coli serotype 01). Reference to OIC herein encompasses the formula OIC and the formula OIC, both identified above.
  • 02 refers to the 02 antigen of E. coli (E. coli serotype 02). Reference to 02 herein encompasses the formula 02 and the formula 02', both identified above.
  • 06 refers to the 06 antigen of E. coli (E. coli serotype 06). Reference to 06 herein encompasses the formula 06 and the formula 06', both identified above.
  • Bioconjugate refers to conjugate between a protein (e.g., a carrier protein) and an antigen, e.g., an O antigen (e.g., 025B) prepared in a host cell background, wherein host cell machinery links the antigen to the protein (e.g., N-links).
  • an antigen e.g., an O antigen (e.g., 025B) prepared in a host cell background, wherein host cell machinery links the antigen to the protein (e.g., N-links).
  • Glycoconjugates include bioconjugates, as well as sugar antigen (e.g., oligo- and
  • polysaccharides)-protein conjugates prepared by other means, e.g., by chemical linkage of the protein and sugar antigen.
  • the term "effective amount,” in the context of administering a therapy (e.g., a composition described herein) to a subject refers to the amount of a therapy which has a prophylactic and/or therapeutic effect(s).
  • an "effective amount” refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an ExPEC infection or symptom associated therewith; (ii) reduce the duration of an ExPEC infection or symptom associated therewith; (iii) prevent the progression of an ExPEC infection or symptom associated therewith; (iv) cause regression of an ExPEC infection or symptom associated therewith; (v) prevent the development or onset of an ExPEC infection, or symptom associated therewith; (vi) prevent the recurrence of an ExPEC infection or symptom associated therewith; (vii) reduce organ failure associated with an ExPEC infection; (viii) reduce hospitalization of a subject having an ExPEC infection; (ix) reduce hospitalization length of a subject having an ExPEC infection; (x) increase the survival of a subject with an ExPEC infection; (xi) eliminate an ExPEC infection in a subject; (xii) inhibit
  • the term "in combination,” in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy.
  • the use of the term “in combination” does not restrict the order in which therapies are administered to a subject.
  • a first therapy e.g., a composition described herein
  • a first therapy can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject.
  • a subject refers to an animal (e.g., birds, reptiles, and mammals).
  • a subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human).
  • a subject is a non- human animal.
  • a subject is a farm animal or pet (e.g., a dog, cat, horse, goat, sheep, pig, donkey, or chicken).
  • a subject is a human.
  • a subject is a human infant. In another embodiment, a subject is a human child. In another embodiment, a subject is a human adult. In another embodiment, a subject is an elderly human. In another embodiment, a subject is a premature human infant.
  • the terms "subject” and “patient” may be used herein interchangeably. [0059] As used herein, the term “premature human infant” refers to a human infant born at less than 37 weeks of gestational age.
  • human infant refers to a newborn to 1 year old human.
  • the term "human toddler” refers to a human that is 1 years to 3 years old.
  • human child refers to a human that is 1 year to 18 years old.
  • human adult refers to a human that is 18 years or older.
  • yielderly human refers to a human 65 years or older.
  • Figure 1 Pathway for 025 A biosynthesis. Arrows indicate individual enzymatic conversions, enzyme names are indicated. Nucleotide activated sugars are prepared in the cytoplasm either by enzymes provided in the O antigen cluster or by housekeeping enzymes of the Gram-negative host cell. A glycosylphosphate transferase (WecA) adds D-GlcNAc phosphate to undecaprenyl phosphate (UP), forming GlcNAc-UPP. Specific
  • glycosyltransferases then elongate the UPP-GlcNAc molecule further by adding
  • Figure 2 rfb cluster, structure, and pathway for wzx/wzy-dependent O-antigen synthesis, exemplified by the E. coli 025 A rfb cluster and O antigen.
  • FIG. 3 A. 025 A, 025B, and 016 BRU structures.
  • Black filled genes are genes involved in nucleotide activated monosaccharide biosynthesis, diagonal stripes are predicted glycosyltransferase genes, grey filling indicates BRU processing or transportation genes, and vertical stripes show O-acetyltransferase homologies. Grey boxes indicate homology scores above 25% between the genes; detailed values are indicated. Thin black and grey arrows show annealing locations of typing PCR oligonucleotides for wzy (025A and 025B specific) and the 025B 3' region (025B specific).
  • Figure 4 Serotype distribution from the epidemiology study. E. coli O antigen serotypes identified in samples of community acquired UTI specimens were grouped according to occurrence
  • FIG. Silver and Western stain analysis of LPS from clinical isolates with an 025 positive agglutination phenotype. Strain numbers are indicated above the gel lanes. Individual clones were grown and OD normalized biomass was harvested by centrifugation. Pellets were dissolved in SDS PAGE Lammli buffer and treated with proteinase K to hydrolyze all proteins in the sample. Standard silver staining was applied to the PAGE gel shown in A, and probing of nitrocellulose membranes containing electrotransferred material from identically run gels with commercial 025 agglutination antiserum is shown in B.
  • FIG. 6 2AB HPLC traces of 025 A and 025B samples. 2AB labeled LLO samples from strains upecl38 (dotted line) and upec436 (solid line) were prepared. Peaks were collected and corresponding BRU structures deduced from the MS/MS fragmentation pattern detailed in Fig. 7 are indicated by arrows.
  • Figure 8 Deacetylation of 2AB labeled LLO sample derived from an 025B positive clinical isolate. The 025B specific peak at 50' elution time obtained from 2AB labeled LLOs of a clinical isolate with the 025B genotype was collected, and analyzed by normal phase HPLC after treatment with (solid line) or without (dotted line) NaOH for hydrolysis of ND Cal's Special Patent Program O-acetyl groups. [0073] Figure 9. Monosaccharide composition analysis of 025A and 025B bioconjugates. 025 bioconjugates were produced, purified, and processed for monosaccharide composition analysis. C18 HPLC traces of samples are shown.
  • 025 A (solid) and 025B (dotted) derived samples are compared to a mix of monosaccharides from commercial sources (Glc, GlcNAc, Rha, FucNAc). The elution times of the monosaccharides are indicated by arrows.
  • FIG. 10 Characterization of 025A bioconjugates. Purified final bulk of 4S-EPA- 025A bioconjugates was analyzed by SDS PAGE and visualized by direct Coomassie staining (C) and Western blotting using either anti-EPA antiserum or anti-025 antiserum.
  • FIG. 11 Characterization of 025B bioconjugates. Purified final bulk of 4S-EPA- 025B bioconjugates was analyzed by SDS PAGE and visualized by direct Coomassie staining (C) and Western blotting using either anti-EPA antiserum or anti-025 antiserum.
  • FIG. 12 01 O antigen genetic biosynthesis and chemical structure.
  • A. The rfb cluster and flanking genes of the OIA strain E. coli G1632 (ACCESSION NO. GU299791) is shown. Black, grey and striped color codes are the same as those for figure 2, described above.
  • FIG. 13 Analysis of LPS from clinical isolates with an 01 positive agglutination phenotype.
  • FIG. 15 01 bioconjugates. Small scale expression test of EPA-Ol glycoprotein by E. coli cells (W3110 Ar 3 ⁇ 4016 : :rfbO 1 AwaaL) transformed with an EPA expression plasmid (pGVXN659) and five different pglB expression plasmids: A, pi 14: expression of non-codon optimized, HA tag containing pglB; B, p939: codon optimized, HA tag containing pglB; C, p970: codon optimized, HA tag removed pglB; D, codon optimized, HA tag containing, natural glycosylation site N534Q removed pglB; and E, codon optimized, HA tag removed, natural glycosylation site N534Q removed pglB.
  • FIG. 06 genetic and chemical structures A. O antigen biosynthesis cluster ⁇ rfb cluster) and flanking genes ofE. coli CFT073 (Genbank AE014075.1). Putative gene functions according to BLAST are indicated and genes specific for 06 O antigen biosynthesis are indicated. B. Chemical structures of reported 06 BRU structures (Jann et al, Carbohydr. Res. 263 (1994) 217-225).
  • FIG. 18 Identification of 06 with branching Glc. 2AB labeled LLO samples from 06 clinical isolates were analyzed by LLO fingerprinting. A. Normal phase HPLC traces from 60' onwards are shown. Extracts were prepared from reference strains
  • FIG. 19 02 O antigen genetic biosynthesis and chemical structures.
  • A. O antigen biosynthesis cluster ⁇ rfb cluster) and flanking genes of strain E. coli G1674 (accession No. GU299792). Black, grey, and striped color codes are as described in previous figures (e.g., Fig. 2).
  • Figure 20 Analysis of LPS from clinical isolates with an 02 positive agglutination phenotype.
  • FIG. 21 OPS analysis from strain W3110 AwaaL ArfbW3110::rfbO2 AwekS. A chromatogram of 2AB labelled LLO analysis by normal phase HPLC is shown.
  • FIG. 22 Recognition of 025 A and 025B LPS by anti-025A and anti-025B MBP antisera in a Western blotting analysis.
  • Two nitrocellulose membranes which were obtained after electrotransfer of LPS samples prepared from upec436 (025A) and upecl38 (025B) and separated by a SDS-PAGE. The loading pattern was identical for both membranes, left lane: 025A LPS from upec438, middle lane: 025B LPS from upec 138.
  • MBP bioconjugates were used for immunization of rabbits. Left panel: anti 025B-MBP antiserum; right panel: anti 025A-MBP antiserum.
  • Figure 24 EPA bioconjugates containing the 01 A, 02, and 06 antigens used in the preclinical study. OPS glycans were produced and purified, and analyzed by SDS PAGE and visualized by Coomassie staining.
  • Figure 25 shows mean ELISA titers obtained with sera from rats immunized with OIA-EPA (Gl), carrier protein alone (G10), TBS (Gl 1), or a tetravalent composition composed of EPA-OIA, 02, 06Glc, and 025B (G12), probed against an ELISA plate coated with OIA-LPS purified from strain upec032.
  • Figure 26 shows mean ELISA titers obtained with sera from rats immunized with 02 -EPA (G4), carrier protein alone (G10), TBS (Gl 1), or a tetravalent composition composed of EPA-OIA, 02, 06Glc, and 025B (G12), probed against an ELISA plate coated with 02 LPS purified from strains CCUG25.
  • Figure 27 shows mean ELISA titers obtained with sera from rats immunized with 06Glc-EPA (G7), carrier protein alone (G10), TBS (Gl 1), or a tetravalent composition composed of EPA-OIA, 02, 06Glc, and 025B (G12), probed against an ELISA plate coated with 06Glc-LPS purified from strain CCUG11309.
  • Figure 28 shows mean ELISA titers obtained with sera from rats immunized with 025B-EPA (G9), carrier protein alone (G10), TBS (Gl 1), or a tetravalent composition composed of 01 A, 02, 06Glc, and 025B (G12), probed against an ELISA plate coated with 025B-LPS purified from strain upecl77.
  • Figure 29 Opsonization indices of sera derived from rats pre-immunization (empty circles) compared to 42 days post-immunization (filled squares) with one priming dose and two booster doses of indicated doses of monovalent vaccine.
  • A 02-EPA immunization
  • B 06-EPA immunization
  • C 025B-EPA immunization.
  • Figure 30 ELISA titers obtained with sera from human subjects vaccinated with a tetravalent vaccine comprising E. coli antigens 01 A, 02, 06Glc, and 025B. A significant increase in the ELISA titers between post (30 days after injection) and pre-injection (day 1) was observed only in the vaccinated groups (*, represents statistical significance).
  • FIG 31 Opsonic Index (OI) obtained with sera from human subjects vaccinated with a tetravalent vaccine comprising E. coli antigens 01 A, 02, 06Glc, and 025B. Immune response as indicated by 01 against placebo and components of the tetravalent vaccine (OIA- EPA (31 A), 02-EPA (3 IB), 06Glc-EPA (31C), and 025B-EPA (3 ID)) before and after injection are depicted. Pre-injection, defined as day 1, is represented by V2 (visit 2), and post-injection, defined as day 30, is represented by V4 (visit 4).
  • OI Opsonic Index
  • Figure 32 ELISA titers (expressed as EC50 values) of sera from vaccinated subjects toward 025A LPS (black bars) and 025B LPS (grey bars), at day 1 (pre- vaccination) and after 30 days (post-vaccination).
  • a statistically significant increase in the ELISA titers between post-injection (30 days after injection) and pre-injection (day 1) was observed for both serotypes: 025A LPS (black bars) and 025B LPS (grey bars).
  • Figure 33 Reactivity of sera from vaccinated subjects toward 025 A (black lines) and 025B (grey lines) expressing E. coli strains. Dotted grey line: serotype 075 strain, a negative control. Figure 33 demonstrates that vaccine-induced serum IgG antibodies from vaccinated subjects strongly respond to 025A and 025B strains.
  • nucleic acids capable of producing 025B in host cells are provided herein.
  • host cells e.g., recombinantly engineered host cells, comprising nucleic acids capable of 025B production.
  • Such host cells can be used to generate bioconjugates comprising 025B linked to a carrier protein, which can be used in, e.g., the formulation of therapeutics (e.g., vaccines).
  • the 025B antigen described herein also is useful in the generation of antibodies, which can be used, e.g., in therapeutic methods such as passive immunization of subjects.
  • compositions comprising 025B, alone or in combination with other E. coli antigens (e.g., 01, 02, and 06 and subserotypes thereof), for use in therapeutic methods, e.g., vaccination of hosts against infection with E. coli (e.g., uropathogenic E. coli).
  • nucleic acids related to 025B production e.g., nucleic acids encoding one or more proteins of an E. coli 025B rfb cluster.
  • nucleic acids encoding one or more proteins of an E. coli 025B rfb cluster.
  • a protein having a specific amino acid sequence can be encoded by multiple different nucleic acids.
  • a nucleic acid provided herein can be altered in such a way that its sequence differs from a sequence provided herein, without affecting the amino acid sequence of the protein encoded by the nucleic acid.
  • coli 025B rfb cluster In a specific embodiment, provided herein is a nucleic acid encoding an E. coli r 3 ⁇ 4(upecl38) gene cluster (SEQ ID NO: 12). In another specific embodiment, provided herein is a nucleic acid encoding a gene cluster that is about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: 12. Upecl38 is an example of an E.coli strain of 025B serotype.
  • nucleic acid encoding an E. coli 025B rfb cluster In another specific embodiment, provided herein is a nucleic acid encoding an E. coli r 3 ⁇ 4(upecl63) gene cluster. In another specific embodiment, provided herein is a nucleic acid encoding a gene cluster that is about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%o, or 99%) identical to or homologous to an E. coli r 3 ⁇ 4(upecl63) gene cluster.
  • nucleic acid encoding an E. coli 025B rfb cluster In a specific embodiment, provided herein is a nucleic acid encoding an E. coli r 3 ⁇ 4(upecl77) gene cluster. In another specific embodiment, provided herein is a nucleic acid encoding a gene cluster that is about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%o, or 99%) identical to or homologous to an E. coli r 3 ⁇ 4(upecl77) gene cluster.
  • nucleic acid encoding proteins of an E. coli 025B rfb cluster are provided herein.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO:l, the rmlB gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: 1.
  • coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO:2, the rmlD gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:2.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO:3, the rmlA gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:3.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO:4, the rmlC gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:4.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO:5, the wzx gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:5.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO:6, the wekA gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:6.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO:7, the wekB gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:7.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO:8, the wzy gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:8.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO:9, the w ⁇ J gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:9.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO: 10, the wbbK gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: 10.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein comprises or consists of SEQ ID NO: 11 , the wbbL gene of the E. coli 025B rfb cluster.
  • a nucleic acid encoding a protein of an E. coli 025B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: 11.
  • proteins encoded by the nucleic acids provided herein are proteins encoded by the nucleic acids provided herein.
  • dTDP-Glucose 4,6- dehydratase encoded by SEQ ID NO: 1.
  • dTDP-6-Deoxy-D-glucose 3,5-epimerase encoded by SEQ ID NO:2.
  • Glucose- 1 -phosphate thymidylyltransferase encoded by SEQ ID NO:3.
  • provided herein is dTDP-4- dehydrorhamnose 3,5-epimerase, encoded by SEQ ID NO:4.
  • SEQ ID NO:4 dTDP-4- dehydrorhamnose 3,5-epimerase
  • provided herein is O antigen flippase, encoded by SEQ ID NO:5.
  • dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP a-1,3- rhamnosyltransferase encoded by SEQ ID NO:6.
  • UDP-Glc:Glc-Rha(Ac)-GlcNAc-UPP ⁇ -1,6- glucosyltransferase encoded by SEQ ID NO:7.
  • O antigen polymerase encoded by SEQ ID NO:8.
  • provided herein is O-acetyl transferase, encoded by SEQ ID NO:9.
  • UDP- Glc:Rha-GlcNAc-UPP a-1,3- glucosyltransferase encoded by SEQ ID NO: 10.
  • dTDP-Rha:GlcNAc-UPP a- 1,3- rhamnosyltransferase encoded by SEQ ID NO: 11.
  • E. coli antigens of the 025, 01, 02, and 06 serotypes are provided herein.
  • provided herein is an isolated O antigen from E. coli strain upecl38. In another specific embodiment, provided herein is an isolated O antigen from E. coli strain upecl63. In another specific embodiment, provided herein is an isolated O antigen fromE. coli strain upecl77.
  • E. coli 025B antigen of Formula 025B is provided herein.
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • n of at least 80% of the macro molecules in the population is between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • n of at least 80% of the macro molecules in the population is between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • E. coli antigens useful in the compositions described herein include 025A, as well as 01, 02, and 06 antigens, and subserotypes thereof.
  • an 025A antigen (e.g., in isolated form or as part of a bioconjugate) is used in a composition provided herein (e.g., in combination with an 025B antigen (or bioconjugate comprising an 025B antigen)).
  • the 025A antigen is Formula 025A:
  • the 025 A antigen is Formula 025 A':
  • an 01 A antigen (e.g., in isolated form or as part of a bioconjugate) is used in a composition provided herein (e.g., in combination with an 025B antigen (or bioconjugate comprising an 025B antigen)).
  • the 01 A antigen is Formula 01 A:
  • the 01 A antigen is Formula 01A':
  • an OIB antigen (e.g., in isolated form or as part of a bioconjugate) is used in a composition provided herein (e.g., in combination with an 025B antigen (or bioconjugate comprising an 025B antigen)).
  • the OIB antigen is Formula OIB:
  • the OIB antigen is Formula OIB':
  • an OIC antigen e.g., in isolated form or as part of a bioconjugate
  • a composition provided herein e.g., in combination with an 025B antigen (or bioconjugate comprising an 025B antigen)
  • the OIC antigen is Formula OIC:
  • the OIC antigen is Formula OIC:
  • an 02 antigen e.g., in isolated form or as part of a bioconjugate
  • a composition provided herein e.g., in combination with an 025B antigen (or bioconjugate comprising an 025B antigen)
  • the 02 antigen is Formula 02:
  • the 02 antigen is Formula 02':
  • an 06 antigen e.g., in isolated form or as part of a bioconjugate
  • a composition provided herein e.g., in combination with an 025B antigen (or bioconjugate comprising an 025B antigen)
  • the 06 antigen is Formula 06K2 also referred to herein as 06Glc :
  • the 06 antigen is Formula 06K2' (also referred to herein as 06Glc'):
  • the 06 antigen is Formula 06K54 (also referred to herein as 06GlcNA
  • the 06 antigen is Formula 06K54' (also referred to herein as 06GlcNAc'):
  • host cells e.g., prokaryotic host cells, capable of producing E. coli O antigens and bioconjugates comprising such E. coli O antigens.
  • prokaryotic host cells capable of producing E. coli O antigens and bioconjugates comprising such E. coli O antigens.
  • the host cells provided herein comprise (e.g., naturally or through genetic engineering) one or more of the nucleic acids described herein. See Section 5.1.
  • the host cells provided herein produce one or more of the E. coli O antigens described herein, and/or produce bioconjugates comprising one or more of the E. coli O antigens described herein. See Section 5.2.
  • a prokaryotic host cell comprising nucleic acids encoding enzymes (e.g., glycosyltransferases) capable of producing the novel polysaccharide disclosed herein, E. coli 025B.
  • host cells comprising nucleic acids encoding enzymes (e.g., glycosyltransferases) capable of producing other E. coli antigens, e.g., 025 A, 01, 02, and 06, and subserotypes thereof (see Section 5.2).
  • the host cells provided herein may naturally express nucleic acids capable of producing of an O antigen of interest, or the host cells may be made to express such nucleic acids, i.e., in certain embodiments said nucleic acids are heterologous to the host cells and introduced into the host cells using genetic approaches known in the art.
  • the host cells provided herein comprise nucleic acids encoding additional enzymes active in the N- glycosylation of proteins, e.g., the host cell provided herein can further comprise a nucleic acid encoding an oligosaccharyl transferase or nucleic acids encoding other
  • the host cells provided herein comprise a nucleic acid encoding a carrier protein, e.g., a protein to which oligo- and polysaccharides can be attached to form a bioconjugate. See, e.g., Section 5.3.2 for a description of carrier proteins and Section 5.4 for a description of bioconjugates.
  • the host cell is E. coli.
  • Upecl38 is an E. coli strain identified herein as belonging to the 025B serotype, and the rfb gene cluster of the strain (and strains of the 025B serotype in general) has been identified herein for the first time as comprising genes that produce a novel E. coli polysaccharide, 025B.
  • a prokaryotic host cell comprising an E. coli rfb(upecl38) gene cluster (SEQ ID NO: 12), or a gene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: 12.
  • the E. coli rfb(upecl38) gene cluster (SEQ ID NO: 12) is introduced into the host cell by genetic manipulation (e.g., the gene cluster is expressed on a plasmid or plasmids or integrated into the host cell genome (see, e.g., International Patent application No. PCT/EP2013/068737)).
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14) or a carrier protein comprising a consensus sequence or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • some or all of the genes of the rfb cluster are heterologous to the host cell.
  • said oligosaccharyl transferase is heterologous to the host cell.
  • said carrier protein is heterologous to the host cell.
  • the host cell is E. coli.
  • Upecl63 is an E. coli strain identified herein as belonging to the 025B serotype, and the rfb gene cluster of the strain (and strains of the 025B serotype in general) has been identified herein for the first time as comprising genes that produce a novel E. coli polysaccharide, 025B.
  • a prokaryotic host cell comprising an E. coli rfb(upecl63) gene cluster, or a gene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to an E. coli rfb(upecl63) gene cluster.
  • the E. coli rfb(upecl63) gene cluster is introduced into the host cell by genetic manipulation (e.g., the gene cluster is expressed on a plasmid or plasmids or integrated into the host cell genome (see, e.g., International Patent application No. PCT/EP2013/068737)).
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X- Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14) or a carrier protein comprising a consensus sequence or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • some or all of the genes of the rfb cluster are heterologous to the host cell.
  • said oligosaccharyl transferase is heterologous to the host cell.
  • said carrier protein is heterologous to the host cell.
  • the host cell is E. coli.
  • Upecl77 is an E. coli strain identified herein as belonging to the 025B serotype, and the rfb gene cluster of the strain (and strains of the 025B serotype in general) has been identified herein for the first time as comprising genes that produce a novel E. coli polysaccharide, 025B.
  • a prokaryotic host cell comprising an E. coli rfb(upecl77) gene cluster, or a gene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to an E. coli rfb(upecl77) gene cluster.
  • the E. coli rfb(upecl77) gene cluster is introduced into the host cell by genetic manipulation (e.g., the gene cluster is expressed on a plasmid or plasmids or integrated into the host cell genome (see, e.g., International Patent application No. PCT/EP2013/068737)).
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X- Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14) or a carrier protein comprising a consensus sequence or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • some or all of the genes of the rfb cluster are heterologous to the host cell.
  • said oligosaccharyl transferase is heterologous to the host cell.
  • said carrier protein is heterologous to the host cell.
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • said host cell comprises rmlB, rmlD, rmlA, rmlC, wzx, wekA, wekB, wzy, wbbJ, wbbK, and/or wbbL.
  • Such host cells can be engineered using recombinant approaches to comprise one or more plasmids comprising the rmlB, rmlD, rmlA, rmlC, wzx, wekA, wekB, wzy, wbbJ, wbbK, and/or wbbL genes.
  • said one or more plasmids is integrated into the host cell genome.
  • said rmlB comprises or consists of SEQ ID NO: l, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: 1.
  • said rmlD comprises or consists of SEQ ID NO:2, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:2.
  • said rmlA comprises or consists of SEQ ID NO:3, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:3.
  • said rmlC comprises or consists of SEQ ID NO:4, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:4.
  • said wzx comprises or consists of SEQ ID NO:5, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:5.
  • said wekA comprises or consists of SEQ ID NO:6, or is 75%, 80%>, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:6.
  • said wekB comprises or consists of SEQ ID NO:7, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:7.
  • said wzy comprises or consists of SEQ ID NO:8, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:8.
  • said wbbJ comprises or consists of SEQ ID NO:9, or is 75%, 80%>, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:9.
  • said wbbK comprises or consists of SEQ ID NO: 10, or is 75%, 80%>, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: 10.
  • said wbbL comprises or consists of SEQ ID NO:l 1, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: l 1.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14) or a carrier protein comprising a consensus sequence or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • some or all of the genes, rmlB, rmlD, rmlA, rmlC, wzx, wekA, wekB, wzy, wbbJ, wbbK, and wbbL, are heterologous to the host cell.
  • said oligosaccharyl transferase is heterologous to the host cell.
  • said carrier protein is heterologous to the host cell.
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • said host cell comprises one, two, three, four, or more, e.g.
  • rmlB SEQ ID NO: l
  • rmlD SEQ ID NO:2
  • rmlA SEQ ID NO:3
  • rmlC SEQ ID NO:4
  • wzx SEQ ID NO:5
  • wekA SEQ ID NO:6
  • wekB SEQ ID NO:7
  • wzy SEQ ID NO:8
  • wbbJ SEQ ID NO:9
  • wbbK SEQ ID NO: 10
  • wbbL SEQ ID NO: l 1).
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of rmlB (SEQ ID NO: 1).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes a dTDP-Glucose 4,6-dehydratase, e.g., a dTDP-Glucose 4,6- dehydratase encoded by rmlB.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of rmlD (SEQ ID NO:2).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes a dTDP-6-Deoxy-D-glucose 3,5-epimerase, e.g., a dTDP-6- Deoxy-D-glucose 3,5-epimerase encoded by rmlD.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X- Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of rmlA (SEQ ID NO:3).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes a Glucose- 1 -phosphate thymidylyltransferase, e.g., a Glucose- 1 -phosphate thymidylyltransferase encoded by rmlA.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X- Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of rmlC (SEQ ID NO:4).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes a dTDP-4-dehydrorhamnose 3,5-epimerase, e.g., a dTDP-4- dehydrorhamnose 3,5-epimerase encoded by rmlC.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X- Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of wzx (SEQ ID NO:5).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes an O antigen flippase, e.g., an O antigen flippase encoded by wzx.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of wekA (SEQ ID NO: 6).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes a rhamnosyltransferase, e.g., an dTDP-Rha:Glc-Rha(Ac)- GlcNAc-UPP a- 1,3- rhamnosyltransferase encoded by wekA.
  • a rhamnosyltransferase e.g., an dTDP-Rha:Glc-Rha(Ac)- GlcNAc-UPP a- 1,3- rhamnosyltransferase encoded by wekA.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise nucleic acids that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X- Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of wekB (SEQ ID NO: 7).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes a wekB glucosyltransferase, e.g., a UDP-Glc:Glc-Rha(Ac)- GlcNAc-UPP ⁇ -1,6- glucosyltransferase encoded by wekB.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of wzy (SEQ ID NO: 8).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes an O antigen polymerase, e.g., an O antigen polymerase encoded by wzy.
  • O antigen polymerase e.g., an O antigen polymerase encoded by wzy.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn- Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of wbbJ (SEQ ID NO: 9).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes an O-acetyl transferase, e.g., an O-acetyl transferase encoded by wbbJ.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of wbbK (SEQ ID NO: 10).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes a glucosyltransferase, e.g., a UDP-Glc:Rha-GlcNAc-UPP a- 1,3- glucosyltransferase encoded by wbbK.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen), wherein said host cell naturally comprises or is engineered to comprise the nucleic acid sequence of wbbL (SEQ ID NO: 11).
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • coli 025B antigen wherein said host cell naturally comprises or is engineered to comprise a nucleic sequence that encodes a rhamnosyl transferase, e.g., a dTDP-Rha:GlcNAc-UPP a- 1,3- rhamnosyltransferase encoded by wbbL.
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO:15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • a prokaryotic host cell capable of producing 025B, and/or an 025B bioconjugate (i.e., a carrier protein linked to the E. coli 025B antigen)
  • said host cell naturally comprises or is engineered to comprise at least one, two, three, four or more, e.g.
  • nucleic acid sequence that is at least 80%>, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: l;
  • nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), or 100%) identical to SEQ ID NO:2;
  • nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
  • said host cell has been engineered to comprise each of said sequences, i.e., said sequences are heterologous to said host cell.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • said host cell comprises at least two of (i) wbbJ (SEQ ID NO: 9) or a nucleic acid that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:9; (ii) wbbK (SEQ ID NO: 10) or a nucleic acid that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: 10; and/or (iii) wbbL (SEQ ID NO: l 1) or a nucleic acid that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 9
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • said host cell comprises each of (i) wbbJ (SEQ ID NO:9) or a nucleic acid that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:9;
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • said host cell comprises (i) dTDP-Glucose 4,6-dehydratase; (ii) dTDP-6-Deoxy-D-glucose 3,5-epimerase;
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14) or a carrier protein comprising a consensus sequence or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • dTDP-Glucose 4,6-dehydratase (i) dTDP-6-Deoxy-D-glucose 3,5-epimerase; (iii) Glucose- 1 -phosphate thymidylyltransferase; (iv) dTDP-4-dehydrorhamnose 3,5-epimerase; (v) O antigen flippase; (vi) dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP a- 1,3- rhamnosyltransferase; (vii) UDP-Glc:Glc- Rha(Ac)-GlcNAc-UPP ⁇ -1,6- glucosyltransferase (viii) O antigen polymerase; (ix) O-acetyl transferase; (x) UDP-Glc:Rha-GlcNAc-UPP
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • said host cell comprises enzymes capable of synthesizing E. coli 025A (see, e.g., Figure 3).
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • said host cell comprises enzymes capable of synthesizing E. coli 01 (see, e.g., Figure 12).
  • a host cell that produces E. coli 01 A.
  • a host cell that produces E. coli OIB In a specific embodiment, provided herein is a host cell that produces E. coli OIC.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • said host cell comprises enzymes capable of synthesizing E. coli 02 (see, e.g., Figure 19).
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell
  • said host cell comprises enzymes capable of synthesizing E. coli 06 (see, e.g., Figure 17).
  • a host cell that produces E. coli 06 comprising a branching Glc monosaccharide or an 06 antigen comprising a branching GlcNAc monosaccharide.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • a prokaryotic host cell e.g., a recombinantly engineered prokaryotic host cell capable of producing more than one type of E. coli O antigen.
  • a host cell capable of producing at least two of the following: 025B, 025A, 01 (e.g., OIA, OIB, OIC), 02, and 06.
  • a host cell capable of producing 025B and one or more of 025A, 01 (e.g., OIA, OIB, OIC), 02, and 06.
  • the prokaryotic host cell comprises a nucleic acid sequence encoding an oligosaccharyl transferase.
  • the prokaryotic host cell further comprises a nucleic acid sequence encoding a carrier protein comprising a consensus sequence Asn-X- Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987).
  • the host cell is E. coli.
  • Any host cells known to those of skill in the art can be used to produce the E. coli O antigens described herein (e.g., 025B) and bioconjugates comprising the E. coli O antigens described herein (e.g., 025B), including archea, prokaryotic host cells, and eukaryotic host cells.
  • Exemplary prokaryotic host cells for use in production of the E. coli O antigens described herein and bioconjugates comprising the E. coli O antigens described herein include, without limitation, Escherichia species, Shigella species, Klebsiella species, Xhantomonas species, Salmonella species, Yersinia species, Lactococcus species,
  • Lactobacillus species Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species.
  • the host cell used to produce the E. coli O antigens described herein and bioconjugates comprising the E. coli O antigens described herein is E. coli.
  • the host cells used to produce the E. coli O antigens and bioconjugates described herein are engineered to comprise heterologous nucleic acids, e.g., heterologous nucleic acids that encode one or more carrier proteins and/or heterologous nucleic acids that encode one or more proteins, e.g., genes encoding one or more proteins.
  • heterologous nucleic acids that encode proteins involved in glycosylation pathways e.g., prokaryotic and/or eukaryotic glycosylation pathways
  • Such nucleic acids may encode proteins including, without limitation, oligosaccharyl transferases and/or glycosyltransferases.
  • Heterologous nucleic acids e.g., nucleic acids that encode carrier proteins and/or nucleic acids that encode other proteins, e.g., proteins involved in glycosylation
  • heterologous nucleic acids are introduced into the host cells described herein using a plasmid, e.g., the heterologous nucleic acids are expressed in the host cells by a plasmid (e.g., an expression vector).
  • a plasmid e.g., an expression vector
  • heterologous nucleic acids are introduced into the host cells described herein using the method of insertion described in International Patent application No.
  • additional modifications may be introduced (e.g., using recombinant techniques) into the host cells described herein.
  • host cell nucleic acids e.g., genes
  • proteins that form part of a possibly competing or interfering glycosylation pathway e.g., compete or interfere with one or more heterologous genes involved in glycosylation that are recombinantly introduced into the host cell
  • host cell background e.g., a sequence that makes them
  • inactive/dysfunctional i.e., the host cell nucleic acids that are deleted/modified do not encode a functional protein or do not encode a protein whatsoever.
  • nucleic acids when nucleic acids are deleted from the genome of the host cells provided herein, they are replaced by a desirable sequence, e.g., a sequence that is useful for glycoprotein production.
  • Exemplary genes that can be deleted in host cells include genes of host cells involved in glyco lipid biosynthesis, such as waaL (see, e.g., Feldman et al, 2005, PNAS USA 102:3016-3021), the lipid A core biosynthesis cluster (waa), galactose cluster (gal), arabinose cluster (ara), colonic acid cluster ( vc), capsular polysaccharide cluster, undecaprenol-p biosynthesis genes (e.g.
  • the host cells described herein are modified such that they do not produce any O antigens other than a desired O antigen from an ExPEC, e.g., 025B.
  • one or more of the waaL gene, gtrA gene, gtrB gene, gtrS gene, or the rfb gene cluster is deleted or functionally inactivated from the genome of a prokaryotic host cell provided herein.
  • a host cell used herein is E.
  • a host cell used herein is E. coli that produces 025B antigen, wherein the waaL gene and gtrS gene are deleted or functionally inactivated from the genome of the host cell.
  • the modified host cells provided herein can be used for protein glycosylation.
  • Protein glycosylation may be designed to produce bioconjugates for use in vaccine formulations, e.g., vaccines that contain E. coli polysaccharide antigen(s), e.g., 025 (e.g., 025B), 01, 02, and 06. 5.3.2 Carrier Proteins
  • any carrier protein suitable for use in the production of conjugate vaccines can be used herein, e.g., nucleic acids encoding the carrier protein can be introduced into a host provided herein for the production of a bioconjugate comprising a carrier protein linked to an ExPEC antigen (e.g., 025B).
  • exemplary carrier proteins include, without limitation, detoxified Exotoxin A of P.
  • aeruginosa EPA; see, e.g., Ihssen, et al, (2010) Microbial cell factories 9, 61), CRM 197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, E. coli FimH, E. coli FimHC, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli Sat protein, the passenger domain of E.
  • MBP maltose binding protein
  • Diphtheria toxoid Tetanus toxoid
  • detoxified hemolysin A of S. aureus clumping factor A
  • clumping factor B E. coli
  • the carrier proteins used in the generation of the bioconjugates described herein are modified, e.g., modified in such a way that the protein is less toxic and/or more susceptible to glycosylation.
  • the carrier proteins used in the generation of the bioconjugates described herein are modified such that the number of glycosylation sites in the carrier proteins is maximized in a manner that allows for lower concentrations of the protein to be administered, e.g., in an immunogenic composition, in its bioconjugate form.
  • the carrier proteins described herein are modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosylation sites than would normally be associated with the carrier protein (e.g., relative to the number of glycosylation sites associated with the carrier protein in its native/natural, e.g., "wild-type" state).
  • introduction of glycosylation sites is accomplished by insertion of
  • glycosylation consensus sequences e.g., Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987)) anywhere in the primary structure of the protein.
  • glycosylation sites can be accomplished by, e.g., adding new amino acids to the primary structure of the protein (i.e., the glycosylation sites are added, in full or in part), or by mutating existing amino acids in the protein in order to generate the glycosylation sites (i.e., amino acids are not added to the protein, but selected amino acids of the protein are mutated so as to form glycosylation sites).
  • new amino acids i.e., the glycosylation sites are added, in full or in part
  • mutating existing amino acids in the protein i.e., amino acids are not added to the protein, but selected amino acids of the protein are mutated so as to form glycosylation sites.
  • amino acid sequence of a protein can be readily modified using approaches known in the art, e.g., recombinant approaches that include modification of the nucleic acid sequence encoding the protein.
  • glycosylation consensus sequences are introduced into specific regions of the carrier protein, e.g., surface structures of the protein, at the N or C termini of the protein, and/or in loops that are stabilized by disulfide bridges at the base of the protein.
  • the classical 5 amino acid glycosylation consensus sequence may be extended by lysine residues for more efficient glycosylation, and thus the inserted consensus sequence may encode 5, 6, or 7 amino acids that should be inserted or that replace acceptor protein amino acids.
  • a carrier protein is detoxified EPA comprising 4 consensus glycosylation sequences Asp/Glu-X-Asn-Z-Ser/Thr (SEQ ID NO: 15), and has an amino acid sequence as provided in SEQ ID NO: 13.
  • the carrier proteins used in the generation of the bioconjugates described herein comprise a "tag," i.e., a sequence of amino acids that allows for the isolation and/or identification of the carrier protein.
  • a tag i.e., a sequence of amino acids that allows for the isolation and/or identification of the carrier protein.
  • adding a tag to a carrier protein described herein can be useful in the purification of that protein and, hence, the purification of conjugate vaccines comprising the tagged carrier protein.
  • Exemplary tags that can be used herein include, without limitation, histidine (HIS) tags (e.g., hexa histidine- tag, or 6XHis-Tag), FLAG-TAG, and HA tags.
  • the tags used herein are removable, e.g., removal by chemical agents or by enzymatic means, once they are no longer needed, e.g., after the protein has been purified.
  • the carrier proteins described herein comprise a signal sequence that targets the carrier protein to the periplasmic space of the host cell that expresses the carrier protein.
  • the signal sequence is from E. coli DsbA, E. coli outer membrane porin A (OmpA), E. coli maltose binding protein (MalE), Erwinia carotovorans pectate lyase (PelB), Flgl, NikA, or Bacillus sp. endoxylanase (XynA), heat labile E. coli enterotoxin LTIIb, Bacillus endoxylanase XynA, or E. coli flagellin (Flgl).
  • the host cells provided herein comprise, and/or can be modified to comprise, nucleic acids that encode genetic machinery (e.g., glycosyltransferases) capable of producing O antigens from ExPEC, e.g., the 025 (e.g., 025B), 01, 02, and/or 06 antigens. See Section 5.1. Glycosyltransferases
  • the host cells provided herein comprise nucleic acids that encode
  • glycosyltransferases capable of producing ExPEC O antigens, e.g., an O antigen from E. coli of serotype 025 (e.g., 025A or 025B, see Figure 3B), 01 (see Figure 12), 02 (see Figure 19), and 06 (e.g., an 06 serotype producing an 06 antigen comprising a branching Glc monosaccharide or an 06 antigen comprising a branching GlcNAc monosaccharide, see Figure 17).
  • O antigen from E. coli of serotype 025 e.g., 025A or 025B, see Figure 3B
  • 01 see Figure 12
  • 02 see Figure 19
  • 06 e.g., an 06 serotype producing an 06 antigen comprising a branching Glc monosaccharide or an 06 antigen comprising a branching GlcNAc monosaccharide, see Figure 17.
  • Exemplary nucleic acids are described in Section 5.1.
  • nucleic acids that encode glycosyltransferases capable of producing an ExPEC O antigen are naturally expressed by the host cells provided herein (e.g., the nucleic acids are present in the "wild-type" background of the host cell). In certain embodiments, some or all of the nucleic acids that encode glycosyltransferases capable of producing an ExPEC O antigen are not naturally expressed by the host cells provided herein, i.e., some or all of the nucleic acids are heterologous to the host cell.
  • Host cells can be engineered to comprise specific nucleic acids, e.g., the nucleic acids described in Section 5.1, using methods known in the art, e.g., the methods described in Section 5.3.
  • the host cells provided herein comprise nucleic acids that encode glycosyltransferases capable of producing an E. coli O antigen of the 025B serotype, i.e., an 025B antigen described herein.
  • said nucleic acids encode the rfb cluster from upecl38 (SEQ ID NO: 12), or a gene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to or homologous to SEQ ID NO: 12.
  • said nucleic acids encode the rfb cluster from upecl63, or a gene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to or homologous to the rfb cluster from upecl63.
  • said nucleic acids encode the rfb cluster from upecl77, or a gene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to or homologous to the rfb cluster from upecl77.
  • glycosyltransferases capable of producing an E. coli O antigen of the 025B serotype are genes of an 025B serotype, wherein said genes are rmlB (SEQ ID NO: l), rmlD (SEQ ID NO:2) rmlA (SEQ ID NO:3), rmlC (SEQ ID NO:4), wzx (SEQ ID NO:5), wekA (SEQ ID NO:6), wekB (SEQ ID NO:7), wzy (SEQ ID NO:8), wbbJ (SEQ ID NO:9), wbbK (SEQ ID NO: 10), and wbbL (SEQ ID NO: l 1). See tables 3 and 9.
  • the host cells provided herein comprise nucleic acids that encode proteins (e.g., glycosyltransferases) capable of producing an E. coli O antigen of the 025A serotype, i.e., an 025A antigen described herein.
  • coli O antigen of the 025A serotype are genes of an 025 serotype, wherein said genes are rmlB, rmlD, rmlA, rmlC, wzx, wekA, wekB, wekC, wzy, fnlA, fnlB, fnlC, wbuB, and/or wbuC. See Wang, et al. (2010) J Clin Microbiol 48, 2066-2074; GenBank GU014554; and Table 2.
  • the host cells provided herein comprise nucleic acids that encode glycosyltransferases capable of producing an O antigen E. coli of the 02 serotype.
  • said nucleic acids that encode glycosyltransferases capable of producing an E. coli O antigen of the 02 serotype are genes of an 02 serotype, wherein said genes are rmlB, rmlD, rmlA, rmlC, wzx, wekP, wekQ, wekR, wzy, fdtA, fdtB, and/or fdtC. See Li, et al, (2010) J Microbiol Methods 82, 71-77; Fratamico, et al, 2010, Canadian Journal of Microbiology 56, 308-316; and Table 5.
  • the host cells provided herein comprise nucleic acids that encode glycosyltransferases capable of producing an O antigen E. coli of the 06 serotype. See Welch et al, 2002, PNAS USA 99(26): 17020-17024; Jann et al, Carbohydr. Res. 263 (1994) 217-225, and Jansson et al, Carbohydr. Res. 131 (1984) 277-283.
  • said 06 serotype comprises a branched Glc monosaccharide.
  • the host cells provided herein comprise nucleic acids that encode glycosyltransferases capable of producing an O antigen E. coli of the 01 serotype.
  • said 01 serotype is 01 A.
  • said nucleic acids that encode glycosyltransferases capable of producing an E. coli O antigen of the 01 serotype are genes of an 01 serotype, wherein said genes are rmlB, rmlD, rmlA, rmlC, wzx, mnaA, wekM, wzy, wekN, and/or wekO.
  • Oligosaccharyl transferases transfer lipid-linked oligosaccharides to asparagine residues of nascent polypeptide chains that comprise an N-glycoxylation consensus motif, e.g., Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15) (see WO 2006/119987). See, e.g., WO 2003/074687 and WO 2006/119987, the disclosures of which are herein incorporated by reference in their entirety.
  • the host cells provided herein comprise a nucleic acid that encodes an oligosaccharyl transferase.
  • the nucleic acid that encodes an oligosaccharyl transferase can be native to the host cell, or can be introduced into the host cell using genetic approaches, as described above.
  • the oligosaccharyl transferase can be from any source known in the art. In a specific embodiment, the oligosaccharyl transferase is an
  • the oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter jejuni (i.e., pglB; see, e.g., Wacker et al, 2002, Science 298: 1790-1793; see also, e.g., NCBI Gene ID: 3231775, UniProt Accession No. 086154).
  • the oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter jejuni (i.e., pglB; see, e.g., Wacker et al, 2002, Science 298: 1790-1793; see also, e.g., NCBI Gene ID: 3231775, UniProt Accession No. 086154).
  • the oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter jejuni (i.e., pglB;
  • oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter lari (see, e.g., NCBI Gene ID: 7410986).
  • the host cells described herein can be used to produce bioconjugates comprising an E. coli O antigen described herein (e.g., 025B; see Section 5.2) linked to a carrier protein.
  • E. coli O antigen described herein e.g., 025B; see Section 5.2
  • Methods of producing such bioconjugates using host cells are known in the art. See, e.g., WO 2003/074687 and WO 2006/119987.
  • glycoconjugates can be prepared by chemical synthesis, i.e., prepared outside of host cells (in vitro).
  • E. coli O antigens described herein e.g., 025B
  • carrier proteins including by means of using activation reactive groups in the
  • Such approaches comprise extraction of antigenic polysaccharides/ oligosaccharides from host cells, purifying the polysaccharides/oligosaccharides, chemically activating the
  • polysaccharides/oligosaccharides conjugating the polysaccharides/ oligosaccharides to a carrier protein.
  • Bioconjugates as described herein, have advantageous properties over
  • glycoconjugates e.g., bioconjugates require less chemicals in manufacture and are more consistent in terms of the final product generated. Thus, bioconjugates are preferred over chemically produced glycoconjugates.
  • bioconjugates comprising a carrier protein linked to an ExPEC O antigen described herein. See Section 5.2.
  • a bio conjugate comprising a carrier protein (e.g., EPA) N-linked to E. coli 025B.
  • bioconjugate comprising a carrier protein (e.g., EPA) linked to a compound of Formula 025B presented below:
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • the carrier protein is N-linked to the O antigen of Formula 025B, i.e., 025B is linked to the Asn residue of a carrier protein comprising the sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 15).
  • bioconjugate comprising a carrier protein (e.g., EPA) linked to a compound of Formula 025B', presented below:
  • n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
  • the carrier protein is N- linked to the O antigen of Formula 025B'.
  • a bioconjugate comprising a carrier protein (e.g., EPA) linked to an 025A antigen.
  • said 025A antigen comprises the formula 025A:
  • a bioconjugate comprising a carrier protein (e.g., EPA) linked to an 01 antigen.
  • said 01 antigen is 01 A, e.g., said antigen comprises the formula 01 A:
  • said 01 antigen is OIB, e.g., said antigen comprises the formul
  • said 01 antigen is OIC, e.g., said antigen comprises the formul
  • a bioconjugate comprising a carrier protein (e.g., EPA) linked to an 02 antigen.
  • EPA carrier protein
  • said 02 antigen comprises the formula 02:
  • a bioconjugate comprising a carrier protein (e.g., EPA) linked to an 06 antigen.
  • said 06 antigen comprises the formula 06Glc:
  • bioconjugates described herein can be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g., ion exchange, anionic exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. See, e.g., Saraswat et al, 2013, Biomed. Res. Int. ID#312709 (p. 1-18); see also the methods described in WO
  • bioconjugates may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • the actual conditions used to purify a particular bioconjugate will depend, in part, on the synthesis strategy (e.g., synthetic production vs. recombinant production) and on factors such as net charge, hydrophobicity, and/or hydrophilicity of the bioconjugate, and will be apparent to those having skill in the art.
  • the 025B antigen described herein (see Section 5.2) and/or bioconjugates comprising the 025B antigen described herein (see Section 5.4) can be used to elicit neutralizing antibodies against ExPEC.
  • the 025B antigen described herein and/or bioconjugates comprising the 025B antigen described herein can be administered to a subject (e.g., a human, mouse, rabbit, rat, guinea pig, etc.) to induce an immune response that includes the production of antibodies.
  • a subject e.g., a human, mouse, rabbit, rat, guinea pig, etc.
  • Such antibodies can be isolated using techniques known to one of skill in the art (e.g., immunoaffinity chromatography, centrifugation, precipitation, etc.).
  • the 025B antigen described herein can be used to screen for antibodies from antibody libraries.
  • isolated 025B can be immobilized to a solid support (e.g., a silica gel, a resin, a derivatized plastic film, a glass bead, cotton, a plastic bead, a polystyrene bead, an alumina gel, or a polysaccharide, a magnetic bead), and screened for binding to antibodies.
  • a solid support e.g., a silica gel, a resin, a derivatized plastic film, a glass bead, cotton, a plastic bead, a polystyrene bead, an alumina gel, or a polysaccharide, a magnetic bead
  • antibodies to be screened may be immobilized to a solid support and screened for binding to 025B.
  • Any screening assay such as a panning assay, ELISA, surface plasmon resonance, or other antibody screening assay known in the art may be used to screen for antibodies that bind to 025B.
  • the antibody library screened may be a commercially available antibody library, an in vitro generated library, or a library obtained by identifying and cloning or isolating antibodies from an individual infected with EXPEC.
  • Antibody libraries may be generated in accordance with methods known in the art. In a particular embodiment, the antibody library is generated by cloning the antibodies and using them in phage display libraries or a phagemid display library.
  • Antibodies identified or elicited using 025B and/or a bioconjugate of 025B can include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds to 025B.
  • the immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2 ) or subclass of immunoglobulin molecule.
  • Antibodies include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide- linked Fvs (sdFv), and anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies elicited or identified using a method described herein), and epitope-binding fragments of any of the above.
  • an antibody elicited or identified using 025B and/or a bioconjugate of 025 B is a human or humanized monoclonal antibody.
  • Antibodies elicited or identified using using 025B and/or a bioconjugate of 025B can be used to monitor the efficacy of a therapy and/or disease progression. Any
  • immunoassay system known in the art may be used for this purpose including, but not limited to, competitive and noncompetitive assay systems using techniques such as
  • radioimmunoassays ELISA (enzyme linked immunosorbent assays), "sandwich"
  • immunoassays precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and Immunoelectrophoresis assays.
  • Antibodies elicited or identified using using 025B and/or a bioconjugate of 025B can be used to detect E. coli 025B strains, for example, from a plurality of E. coli strains and/or to diagnose an infection by an E. coli 025B strain.
  • compositions comprising the host cells described herein. Such compositions can be used in methods for generating the
  • bioconjugates described herein e.g., the compositions can be cultured under conditions suitable for the production of proteins. Subsequently, bioconjugates can be isolated from said compositions using methods known in the art.
  • compositions comprising the host cells provided herein can comprise additional components suitable for maintenance and survival of the host cells described herein, and can additionally comprise additional components required or beneficial to the production of proteins by the host cells, e.g., inducers for inducible promoters, such as arabinose, IPTG. 5.6.2 Compositions Comprising Antigens and/or Bioconjugates
  • compositions comprising one or more of the E. coli O antigens described herein (see Section 5.2) and compositions (e.g., pharmaceutical compositions) comprising one or more of the bioconjugates described herein (see Section 5.4).
  • a composition provided herein comprises one or more of the E. coli O antigens described herein (see Section 5.2).
  • a composition provided herein comprises one or more of the bioconjugates described herein (see Section 5.4).
  • a composition provided herein comprises one or more of the E.
  • compositions described herein are useful in the treatment and prevention of infection of subjects (e.g., human subjects) with extra-intestinal pathogenic E. coli (ExPEC). See Section 5.7.
  • compositions in addition to comprising an E. coli O antigen described herein (see Section 5.2) and/or a bioconjugate described herein (see Section 5.4), the compositions (e.g., pharmaceutical compositions) described herein comprise a
  • pharmaceutically acceptable carrier As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans.
  • carrier as used herein in the context of a pharmaceutically acceptable carrier, refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • composition comprising a carrier protein (e.g., a carrier protein described in Section 5.3.2) linked to an antigen described herein, e.g., an ExPEC O antigen described in Section 5.2.
  • a composition provided herein comprises a carrier protein (e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP) linked to E. coli 025B (see Section 5.2).
  • composition provided herein comprises a carrier protein (e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP) linked to E. coli 025 A (see Section 5.2).
  • a carrier protein e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP
  • E. coli 025 A see Section 5.2.
  • composition provided herein comprises a carrier protein (e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP) linked to E. coli
  • a carrier protein e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP
  • said 01 macromolecule is 01 A. In another specific embodiment, said 01 macromolecule is OIB. In another specific embodiment, said 01 macromolecule is 01C.
  • composition provided herein comprises a carrier protein (e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP) linked to E. coli
  • a carrier protein e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP
  • a composition provided herein comprises a carrier protein (e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP) linked to E. coli 06 (see Section 5.2).
  • a carrier protein e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP
  • said 06 macromolecule is an 06
  • composition e.g., a pharmaceutical composition, comprising (i) an 025 (e.g., 025A or 025B) macromolecule, or a bioconjugate comprising 025 (e.g., 025A or 025B) and (ii) an 01 macromolecule or a bioconjugate comprising 01.
  • said 025 macromolecule is an 025B macromolecule.
  • said 01 macromolecule is 01 A.
  • said 01 macromolecule is OIB.
  • said 01 macromolecule is 01C.
  • composition e.g., a pharmaceutical composition, comprising (i) an 025 (e.g., 025A or 025B) macromolecule, or a bioconjugate comprising 025 (e.g., 025A or 025B) and (ii) an 02 macromolecule or a bioconjugate comprising 02.
  • 025 e.g., 025A or 025B
  • said 025 macromolecule is an 025B macromolecule.
  • composition e.g., a pharmaceutical composition, comprising (i) an 025 (e.g., 025A or 025B) macromolecule, or a bioconjugate comprising 025 (e.g., 025A or 025B) and (ii) an 06 macromolecule (e.g., a 06 macromolecule comprising a branching Glc monosaccharide or a branching GlcNAc monosaccharide) or a bioconjugate comprising 06.
  • 025 macromolecule is an 025B macromolecule.
  • said 06 macromolecule is an 06 macromolecule comprising a branching Glc monosaccharide.
  • composition e.g., a pharmaceutical composition, comprising E. coli 025B (see Section 5.2) or a or a
  • bioconjugate comprising 025 (see Section 5.4) and at least one of the following: (i) E. coli 01 or a bioconjugate comprising 01 (see Sections 5.2 and 5.4); (ii) E. coli 02 or a bioconjugate comprising 02 (see Sections 5.2 and 5.4); and/or (iii) E. coli 06 or a bioconjugate comprising 06 (see Sections 5.2 and 5.4).
  • said 01 is 01A.
  • said 01 is OIB.
  • said 01 is 01 C.
  • said 06 is 06 comprising a branching Glc monosaccharide.
  • composition e.g., a pharmaceutical composition, comprising at least two of the following: (i) an 025 (e.g., 025A or 025B) macromolecule or a bioconjugate comprising 025 (e.g., 025 A or 025B); (ii) an 01 macromolecule or a bioconjugate comprising 01; (iii) an 02 macromolecule or a
  • said 025 macromolecule is an 025B macromolecule.
  • said 01 macromolecule is 01 A.
  • said 01 macromolecule is OIB.
  • said 01 macromolecule is 01C.
  • said 06 macromolecule is an 06 macromolecule comprising a branching Glc monosaccharide.
  • composition e.g., a pharmaceutical composition, comprising a bioconjugate comprising E. coli 025B and a bioconjugate comprising E. coli 01 A.
  • bioconjugates are described in Section 5.4.
  • composition e.g., a pharmaceutical composition, comprising a bioconjugate comprising E. coli 025B and a bioconjugate comprising E. coli OIB.
  • bioconjugates are described in Section 5.4.
  • a composition e.g., a pharmaceutical composition, comprising a bioconjugate comprising E. coli 025B and a bioconjugate comprising E. coli 01C.
  • a bioconjugate comprising E. coli 025B and a bioconjugate comprising E. coli 01C.
  • a composition e.g., a pharmaceutical composition, comprising a bioconjugate comprising E. coli 025B and a bioconjugate comprising E. coli 02.
  • bioconjugates are described in Section 5.4.
  • composition e.g., a pharmaceutical composition, comprising a bioconjugate comprising E. coli 025B and a bioconjugate comprising E. coli 06.
  • bioconjugates are described in Section 5.4.
  • a composition provided herein comprises a carrier protein (e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP) linked to E. coli 025B (see Section 5.2), (ii) a carrier protein (e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP) linked to an E. coli O antigen of the 01 serotype, e.g., 01 A (see Section 5.2), (iii) a carrier protein (e.g., a carrier protein described in Section 5.1.2, e.g., EPA or MBP) linked to an E.
  • a carrier protein e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP
  • a carrier protein e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP linked to an E. coli O antigen of the 06 serotype (see Section 5.2).
  • compositions comprise a carrier protein (e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP) linked to an E. coli O antigen of an E. coli serotype other than 01, 02, 06, or 025.
  • a carrier protein e.g., a carrier protein described in Section 5.3.2, e.g., EPA or MBP
  • E. coli serotypes are described, e.g., in Example 1 and Table 1, below.
  • composition provided herein comprises an 025 (e.g., 025A or 025B) macromolecule.
  • composition provided herein comprises an 01 macromolecule (e.g., 01A, 01B, or 01C).
  • composition provided herein comprises an 02 macromolecule.
  • composition provided herein comprises an 06 macromolecule (e.g., a 06 macromolecule comprising a branching Glc monosaccharide or a branching GlcNAc monosaccharide).
  • 06 macromolecule e.g., a 06 macromolecule comprising a branching Glc monosaccharide or a branching GlcNAc monosaccharide.
  • a composition provided herein comprises an 025 (e.g., 025A or 025B) macromolecule, an 01 macromolecule, an 02 macromolecule, and an 06 macromolecule (e.g., a 06 macromolecule comprising a branching Glc monosaccharide or a branching GlcNAc monosaccharide).
  • 025 e.g., 025A or 025B
  • 01 macromolecule e.g., 025A or 025B
  • an 06 macromolecule e.g., a 06 macromolecule comprising a branching Glc monosaccharide or a branching GlcNAc monosaccharide.
  • said 025 e.g., 025A or 025B
  • compositions provided herein can be used for eliciting an immune response in a host to whom the composition is administered, i.e., are immunogenic.
  • compositions described herein can be used as vaccines against ExPEC infection, or can be used in the treatment of ExPEC infection, and can accordingly be formulated as
  • compositions comprising the bioconjugates and/or macromolecules described herein may comprise any additional components suitable for use in pharmaceutical administration.
  • the compositions described herein are monovalent formulations.
  • the compositions described herein are multivalent formulations, e.g., bivalent, trivalent, and tetravalent formulations.
  • a multivalent formulation comprises more than one bioconjugate or E. coli O antigen described herein. See Sections 5.2 and 5.4 for description of E. coli O antigens and bioconjugates, respectively.
  • a composition described herein is a tetravalent formulation comprising a macromolecule or bioconjugate, wherein said valences are from E. coli O antigens of the 025B, 01 A, 06, and 02 serotypes/subserotypes.
  • compositions described herein additionally comprise a preservative, e.g., the mercury derivative thimerosal.
  • a preservative e.g., the mercury derivative thimerosal.
  • the pharmaceutical compositions described herein comprise 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not comprise a preservative.
  • compositions described herein comprise, or are administered in combination with, an adjuvant.
  • the adjuvant for administration in combination with a composition described herein may be administered before, concomitantly with, or after administration of said composition.
  • the term "adjuvant” refers to a compound that when administered in conjunction with or as part of a composition described herein augments, enhances and/or boosts the immune response to a bioconjugate, but when the compound is administered alone does not generate an immune response to the bioconjugate.
  • the adjuvant generates an immune response to the poly bioconjugate peptide and does not produce an allergy or other adverse reaction.
  • Adjuvants can enhance an immune response by several mechanisms including, e.g., lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages.
  • adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O- acylated monophosphoryl lipid A (MPL) (see United Kingdom Patent GB2220211), MF59 (Novartis), AS03 (Glaxo SmithKline), AS04 (Glaxo SmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No.
  • alum such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate
  • MPL 3 De-O- acylated monophosphoryl lipid A
  • MPL 3 De-O- acylated monophosphoryl lipid A
  • AS03 Gaxo SmithKline
  • AS04 Gaxo SmithKline
  • polysorbate 80 Teween 80; ICL Americas, Inc.
  • imidazopyridine compounds see International Application No.
  • PCT/US2007/064857 published as International Publication No. WO2007/109812
  • imidazoquinoxaline compounds see International Application No. PCT/US2007/064858, published as International Publication No. WO2007/109813
  • saponins such as QS21 (see Kensil et ah, in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540).
  • the adjuvant is Freund's adjuvant (complete or incomplete).
  • adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et ah, N. Engl. J. Med. 336, 86-91 (1997)).
  • immune stimulants such as monophosphoryl lipid A (see Stoute et ah, N. Engl. J. Med. 336, 86-91 (1997)).
  • Another adjuvant is CpG (Bioworld Today, Nov. 15, 1998).
  • compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the compositions described herein may be formulated to be suitable for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration.
  • the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal or pulmonary administration.
  • compositions described herein additionally comprise one or more buffers, e.g., phosphate buffer and sucrose phosphate glutamate buffer. In other embodiments, the compositions described herein do not comprise buffers.
  • compositions described herein additionally comprise one or more salts, e.g., sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts ⁇ e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or a mixture of such aluminum salts).
  • the compositions described herein do not comprise salts.
  • compositions described herein can be included in a container, pack, or dispenser together with instructions for administration.
  • compositions described herein can be stored before use, e.g., the compositions can be stored frozen ⁇ e.g., at about -20°C or at about -70°C); stored in refrigerated conditions ⁇ e.g., at about 4°C); or stored at room temperature.
  • the compositions can be stored frozen ⁇ e.g., at about -20°C or at about -70°C); stored in refrigerated conditions ⁇ e.g., at about 4°C); or stored at room temperature.
  • E. coli O antigen described herein see Section 5.2
  • bioconjugate described herein see Section 5.4
  • composition described herein see Section 5.6.2.
  • compositions described herein are used in the prevention of infection of a subject (e.g., human subjects) by ExPEC, i.e., the compositions described herein are used to vaccinate a subject against ExPEC infection.
  • a subject e.g., human subjects
  • ExPEC ExPEC
  • the compositions described herein are used to vaccinate a subject against ExPEC infection.
  • the compositions described herein are used to vaccinate a subject against ExPEC infection.
  • compositions described herein are used in the treatment of a subject that has been infected by ExPEC.
  • said subject has an ExPEC infection at the time of administration.
  • said subject does not have an ExPEC infection at the time of administration.
  • Also provided herein are methods of inducing the production of opsonophagocytic antibodies against ExPEC in a subject comprising administering to the subject an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2).
  • said subject has an ExPEC infection at the time of administration.
  • said subject does not have an ExPEC infection at the time of administration.
  • a method for preventing an E. coli (e.g., ExPEC) infection in a subject comprises administering to a subject in need thereof an effective amount of a composition described in Section 5.6.2.
  • the methods of preventing ExPEC infection in a subject provided herein result in the induction of an immune response in a subject comprising administering to the subject a of a composition described in Section 5.6.2.
  • One of skill in the art will understand that the methods of inducing an immune response in a subject described herein result in vaccination of the subject against infection by the ExPEC strains whose O antigens are present in the composition(s).
  • an E. coli e.g., ExPEC
  • said method comprises administering to a subject in need thereof an effective amount of a composition described in Section 5.6.2.
  • the immune response induced by an E. coli O antigen described herein is effective to prevent and/or treat an ExPEC infection caused by any serotype, subserotype, or strain of ExPEC.
  • coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is effective to prevent and/or treat an ExPEC infection more than one serotype of ExPEC.
  • the immune response induced by an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is effective to prevent and/or treat an infection caused by E. coli of the 025 serotype.
  • said 025 serotype is 025B.
  • said 025 serotype is 025A.
  • the immune response induced by an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is effective to prevent and/or treat an infection caused by E. coli of the 01 serotype.
  • said 01 serotype is 01 A.
  • said 01 serotype is 01B.
  • said 01 serotype is 01C.
  • the immune response induced by an E. coli O antigen described herein is effective to prevent and/or treat an infection caused by E. coli of the 02 serotype.
  • the immune response induced by an E. coli O antigen described herein is effective to prevent and/or treat an infection caused by E. coli of the 06 serotype.
  • the immune response induced by an E. coli O antigen described herein is effective to prevent and/or treat an infection caused by two or more of the following E. coli serotypes: 025 (e.g., 025B and 025A), 01 (e.g., 01A, 01B, and 01C), 02, and/or 06.
  • the immune response induced by an E. coli O antigen described herein is effective to prevent and/or treat an infection caused by each of the following E. coli serotypes: 025 (e.g., 025B and 025A), 01 (e.g., 01A, 01B, and 01C), 02, and 06.
  • the subject may be administered a single composition described herein, wherein said composition comprises one, two, three, four, or more E. coli antigens described herein. See Section 5.2.
  • the subject may be administered multiple bioconjugates described herein, e.g., a subject may be administered two, three, four, or more bioconjugates described in Section 5.4.
  • the subject in order to treat a subject having an ExPEC infection or immunize a subject against an ExPEC infection, the subject may be administered multiple compositions described herein, e.g., a subject may be administered two, three, four, or more compositions described in Section 5.6.2.
  • the immune response induced in a subject following administration of an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is effective to reduce symptoms resulting from an ExPEC infection.
  • Symptoms of ExPEC infection may vary depending on the nature of the infection and may include, but are not limited to: dysuria, increased urinary frequency or urgency, pyuria, hematuria, back pain, pain while urinating, fever, chills, and/or nausea (e.g., in subjects having a urinary tract infection caused by ExPEC); high fever, headache, stiff neck, nausea, vomiting, seizures, sleepiness, and/or light sensitivity (e.g., in subjects having meningitis caused by ExPEC); fever, increased heart rate, increased respiratory rate, decreased urine output, decreased platelet count, abdominal pain, difficulty breathing, and/or abnormal heart function (e.g., in subjects having sepsis caused by ExPEC).
  • the immune response induced in a subject following administration of an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is effective to reduce the likelihood of hospitalization of a subject suffering from an ExPEC infection.
  • the immune response induced in a subject following administration of an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is effective to reduce the duration of hospitalization of a subject suffering from an ExPEC infection.
  • kits for preventing and/or treating an ExPEC infection in a subject caused by E. coli of the 025B serotype by administering an antibody described herein, i.e., an anti-025B antibody described herein.
  • the neutralizing antibody is a monoclonal antibody.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject in combination with one or more other therapies ⁇ e.g., antibacterial or immunomodulatory therapies).
  • the one or more other therapies may be beneficial in the treatment or prevention of an ExPEC infection or may ameliorate a symptom or condition associated with an ExPEC infection.
  • the one or more other therapies are pain relievers or anti-fever medications.
  • the therapies are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part.
  • anti-bacterial agents known to one of skill in the art may be used in combination with an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2).
  • Non-limiting examples of anti-bacterial agents include Amikacin, Amoxicillin, Amoxicillin- clavulanic acid, Amphothericin-B, Ampicillin, Ampicllin-sulbactam, Apramycin,
  • Cefoperazone Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefpirome, Cefpodoxime, Cefpodoxime-clavulanic acid, Ce odoxime-sulbactam, Cefprozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole, Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin, Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin, Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin, Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin, Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin, Flucloxaci
  • Garenoxacin Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem, Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin, Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem, Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline, Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin, Penicillin V, Piperacillin, Piperacillin-sulbactam, Piperacillin- tazobactam, Rifampicin, Roxythromycin, Sparfloxacin, Spectinomycin, Spiramycin,
  • a combination therapy comprises administration of two or more E. coli O antigens described herein (see Section 5.2), bioconjugates described herein (see Section 5.4), and/or compositions described herein (see Section 5.6.2).
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a na ' ive subject, i.e., a subject that does not have an ExPEC infection or has not previously had an ExPEC infection.
  • a na ' ive subject i.e., a subject that does not have an ExPEC infection or has not previously had an ExPEC infection.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a na ' ive subject that is at risk of acquiring an ExPEC infection.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject who has been diagnosed with an ExPEC infection.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject infected with ExPEC before symptoms manifest or symptoms become severe ⁇ e.g., before the patient requires hospitalization).
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject who has been diagnosed with an UPEC infection.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject suffering from reoccurring urinary tract infections.
  • an E. coli O antigen described herein see Section 5.2
  • a bioconjugate described herein (see Section 5.4)
  • a composition described herein is administered to a subject suffering from reoccurring urinary tract infections.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject suffering from reoccurring urinary tract infections, but is healthy at the moment of treatment.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject having or at risk of acquiring bacteremia or sepsis.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is an animal.
  • the animal is a bird.
  • the animal is a canine.
  • the animal is a feline.
  • the animal is a horse.
  • the animal is a cow.
  • the animal is a mammal, e.g., a horse, swine, mouse, or primate.
  • the subject is a human.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is a human adult.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is a human adult more than 50 years old.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is an elderly human subject.
  • a subject to be administered an E. coli O antigen described herein is a human child.
  • a subject to be administered an E. coli O antigen described herein is a human infant.
  • a subject to be administered an E. coli O antigen described herein is a human infant.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is a premature human infant.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is a human toddler.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered is not an infant of less than 6 months old.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is an individual who is pregnant.
  • an E. coli O antigen described herein see Section 5.2
  • a bioconjugate described herein see Section 5.4
  • a composition described herein see Section 5.6.2
  • a subject to be administered an E. coli O antigen described herein is a woman who has given birth 1, 2, 3, 4, 5, 6, 7, or 8 weeks earlier.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is an individual at increased risk of ExPEC ⁇ e.g., an immunocompromised or immunodeficient individual).
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is an individual in close contact with an individual having or at increased risk of ExPEC infection.
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is a health care worker ⁇ e.g., a doctor or nurse).
  • a subject to be administered an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is immunocompromised (e.g., suffers from HIV infection) or immunosuppressed.
  • a subject to be administered an E. coli O antigen described herein has diabetes.
  • a subject to be administered an E. coli O antigen described herein has multiple sclerosis.
  • a subject to be administered an E. coli O antigen described herein has a condition that requires them to use a catheter.
  • a subject to be administered an E. coli O antigen described herein has a spinal cord injury.
  • the amount of an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) which will be effective in the treatment and/or prevention of an ExPEC infection will depend on the nature of the disease, and can be determined by standard clinical techniques.
  • Administration of the O-antigen, bioconjugate and/or composition can be done via various routes known to the clinician, for instance subcutaneous, parenteral, intravenous, intramuscular, topical, oral, intradermal, transdermal, intranasal, etc. In one embodiment, administration is via intramuscular injection.
  • the precise dosage to be employed in the formulation will also depend on the route of administration, and the seriousness of the infection, and should be decided according to the judgment of the practitioner and each subject's circumstances.
  • effective dosages may also vary depending upon means of administration, target site, physiological state of the patient (including age, body weight, health), whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • an in vitro assay is employed to help identify optimal dosage ranges. See Section 5.8. Effective doses may be extrapolated from dosage response curves derived from in vitro or animal model test systems.
  • exemplary dosages for glycoconjugate based vaccines range from about 0.1 ⁇ g to 400 ⁇ g of carbohydrate per dose. In other embodiments, exemplary dosages for glycoconjugate based vaccines (e.g., compositions comprising bioconjugates) range from about 0.1 ⁇ g to 4000 ⁇ g of protein(s) per dose.
  • an exemplary dosage for a glycoconjugate based vaccine comprises 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ⁇ g of
  • an exemplary dosage for a glycoconjugate based vaccine comprises 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ⁇ g of protein(s) per dose.
  • a dosage for administration to a human corresponds to 0.5 ml containing about 1-10, e.g. about 2-6, e.g. about 4 ⁇ g of polysaccharide for each of the glycoconjugates included.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject once as a single dose.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject as a single dose followed by a second dose 3 to 6 weeks later.
  • booster inoculations may be administered to the subject at 6 to 12 month intervals following the second inoculation.
  • the booster inoculations may utilize a different E. coli O antigen, bioconjugate, or composition.
  • the administration of the same E. coli O antigen, bioconjugate, or composition may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.
  • an E. coli O antigen described herein see Section 5.2
  • a bioconjugate described herein see Section 5.4
  • a composition described herein is administered to a subject as a single dose once per year.
  • an E. coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) is administered to a subject as 2, 3, 4, 5 or more doses 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks apart. In some embodiments, 2, 3, 4, 5 or more doses of an E.
  • coli O antigen described herein (see Section 5.2), a bioconjugate described herein (see Section 5.4), or a composition described herein (see Section 5.6.2) are administered to a subject 2, 3, 4, 5 or 6 weeks apart at a dosage of 0.1 ⁇ g to 0.5 mg, 0.1 ⁇ g to 0.4 mg, 0.1 ⁇ g to 0.3 mg, 0.1 ⁇ g to 0.2 mg, or 0.1 ⁇ g to 0.1 mg carbohydrate content.
  • the E. coli O antigen, bioconjugate, or composition administered is the same each time.
  • the E. coli O antigen, bioconjugate, or composition administered is different each time.
  • the dosage can range from about 0.0001 to 100 mg of antibody per kg of body weight, or from 0.01 to 5 antibody per kg of body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg or in other words, 70 mg or 700 mg or within the range of 70-700 mg, respectively, for a 70 kg patient.
  • An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months for a period of one year or over several years, or over several year- intervals. Intervals can be irregular and altered based on blood levels of antibody in the patient.
  • the ability of the bioconjugates/compositions described herein to generate an immune response in a subject can be assessed using any approach known to those of skill in the art or described herein.
  • the ability of a bioconjugate to generate an immune response in a subject can be assessed by immunizing a subject (e.g., a mouse) or set of subjects with a bioconjugate described herein and immunizing an additional subject (e.g., a mouse) or set of subjects with a control (PBS).
  • a subject e.g., a mouse
  • an additional subject e.g., a mouse
  • PBS control
  • the ability of the ExPEC to cause disease in the subjects or set of subjects can be determined.
  • disease e.g., UTI
  • a bioconjugate(s) or composition thereof described herein suffer less from or do not suffer from disease
  • the bioconjugate is able to generate an immune response in a subject.
  • the ability of a bioconjugate(s) or composition thereof described herein to induce antiserum that cross-reacts with an O antigen from ExPEC can be tested by, e.g., an immunoassay, such as an ELISA.
  • bioconjugates described herein to generate an immune response in a subject can be assessed using a serum bactericidal assay (SBA) or opsonophagocytotic killing assay (OPK), which represents an established and accepted method that has been used to obtain approval of glycoconjugate-based vaccines.
  • SBA serum bactericidal assay
  • OPK opsonophagocytotic killing assay
  • Such assays are well-known in the art and, briefly, comprise the steps of generating and isolating antibodies against a target of interest (e.g., an O antigen, e.g., 025B, of E. coli) by administering to a subject (e.g., a mouse) a compound that elicits such antibodies.
  • a target of interest e.g., an O antigen, e.g., 025B, of E. coli
  • the bactericidal capacity of the antibodies can be assessed by, e.g., culturing the bacteria in question (e.g., E. coli of the relevant serotype) in the presence of said antibodies and complement and - depending on the assay - neutrophilic cells and assaying the ability of the antibodies to kill and/or neutralize the bacteria, e.g., using standard microbiological approaches.
  • the bacteria in question e.g., E. coli of the relevant serotype
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the compositions described herein (see Section 5.6.2), such as one or more E. coli antigens (see Section 5.2) and/or bioconjugates (see Section 5.4) provided herein.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the kits encompassed herein can be used in the above methods of treatment and immunization of subjects.
  • LPS of Gram-negative cells is composed of a lipid A base, modified with a core oligosaccharide providing the attachment for the O antigen.
  • cells were grown in standard LB medium at 37°C for 24 h, and biomass
  • LPS preparation for coating of ELIS A plates [00276] LPS was prepared using a method described by Apicella, (2008) Methods Mol Biol 431 , 3-13, and further purified as described by Perdomo and Montero, (2006) Biotecnologia PVda 23: 124-129.
  • resuspension was passed through a tC 18 Sep-PAK cartridge.
  • the cartridge was conditioned with 10 ml methanol, followed by equilibration with 10 ml 3:48:47 (C:M:W). After loading of the sample, the cartridge was washed with 10 ml 3:48:47 (C:M:W) and eluted with 5 ml methanol and 5 ml 10:10:3 (C:M:W). The combined elutions were dried under N 2 .
  • glyco lipid samples were hydrolyzed by dissolving the dried samples in 2 ml n-propanol:2 M trifluoroacetic acid (1 : 1), heating to 50 °C for 15 min, and then evaporating to dryness under N 2 (Glover, et al, Proc Natl Acad Sci U S A 102(40): 14255-9).
  • the dried samples were once more resuspended in 3:48:47 and passed through a tC18 cartridge, and the flow through was dried under N2. Labeling with 2-AB and glycan cleanup was performed using the paper disk method as described (Bigge, et al, Anal Biochem 230(2): 229-38; Merry, et al, Anal Biochem 304(1): 91-9).
  • Re-N-acetylation of the polysaccharides was performed by incubation in 471 ⁇ 4.5% acetic anhydride in 1 M sodium bicarbonate for two hours on ice. Thereafter, 600 ⁇ of a 5% TFA solution was added and the samples were hydrolyzed for another hour on ice. Purification was performed on an EB20 column using the corresponding buffers EB20 A and B.
  • Bioconjugates were produced by recombinant E. coli cells expressing, via plasmid(s), carrier protein(s) and the oligosaccharyl transferase from C. jejuni (PglB), and OPS from cosmids or chromosomal insertion mutants.
  • EPA Genetically detoxified EPA (Exotoxin A from Pseudomonas aeruginosa containing the mutations L552V, ⁇ 553) was used as a carrier protein, and was modified to comprise 2 or 4 glycosylation sites (referred to herein as 2S-EPA and 4S-EPA, respectively) and a C-terminal HIS Tag, and was expressed from a pBR322 derived, arabinose inducible plasmid (see Ihssen, et al, (2010) Microbial cell factories 9, 61).
  • MBP Maltose binding protein
  • pGVXN579 is a modified pMAL-p2X (New England
  • the PglB protein was expressed from plasmid pEXT21 (an EcoRI/BamHI fragment from pMAFlO (Feldman et al, 2005, PNAS USA 102(8):3016-3021) was cloned into pEXT21 with a C-terminally fused HA-tag. Variants of the expression plasmid are codon optimization (pGVXN939), codon optimization with a deletion of the glycosylation site (pGVXN948), and removed HA-Ttag (pGVXN970) and codon optimization and deleted HA tag (pGVXN971).
  • Clinical isolates were analyzed for their capability to synthetize a certain OPS using agglutination, Western blot, silver staining, LLO fingerprinting, PCR serotyping, or similar technologies that allow identification of the OPS structural characteristics, and also for their antibiotic resistance phenotype. Certain clinical isolates were further
  • chromosomally deleted for the ligase enzyme, WaaL for enhancing OPS availability for protein glycosylation or OPS analysis.
  • the rfb cluster of the laboratory strain W3110 was replaced by the rfb cluster cloned from clinical isolates, and OPS biosynthesis was analyzed.
  • the waaL gene was deleted to enhance efficiency of bioconjugate production.
  • Homologous recombination of large rfb clusters of interest was achieved by using DNA flanking the rfb cluster coding sequence of W3110 of 0.5 to 1.5 kilobases in length and in vivo linearization of the insert DNA from plasmid borne rfb.
  • the resulting strain contained a replaced rfb cluster (with and without antibiotic resistance cassette), i.e. the rfb cluster of W3110 was replaced for a DNA molecule between the gale and gnd genes by the analogous stretch isolated from a clinical E. coli isolate.
  • W3110 strains containing a cosmid encoding the rfb cluster of a given E. coli serotype were used as host strains.
  • W3110 borne genes that interfere with recombinant OPS production were deleted.
  • the gtrABS cluster of W3110 was deleted.
  • homologous recombination according to a published method (Bloor AE, Cranenburgh RM. Appl Environ Microbiol. 2006 Apr;72(4):2520-5.) using homology sequences flanking upstream the gtrA gene and downstream of the gtrS gene.
  • the host strain was transformed with a pglB and a carrier expression plasmid by transformation. See Wacker et al, 2002, Science 298: 1790- 1793.
  • Bioconjugate production was performed by growing host cells and purifying bioconjugates produced in the periplasmic space. Growth was performed either in shake flasks or in an industrial scale fed batch fermentation process.
  • Overhead pressure was maintained at 600 mbar.
  • Product formation was induced with L- Arabinose (0.1%) and/or IPTG (1 mM).
  • feed was initiated by addition of feed medium containing 2.5% Arabinose and IPTG.
  • feed was stopped and harvesting was performed by tangential flow filtration or centrifugation.
  • Bioconjugates were purified by column chromatography. Various
  • bioconjugates mainly IMAC, Q-resin based anionic exchange chromatography (AEC), and size exclusion chromatography (SEC). See, e.g., Saraswat et al, 2013, Biomed. Res. Int. ID#312709 (p. 1-18) and WO 2009/104074 for description of such methods.
  • Bioconjugates containing approximately 8 ug polysaccharide were hydro lyzed for six hours in 104 ⁇ 3 M TFA at 99°C. TFA was removed by evaporation and samples were washed once with 500 ⁇ 2-propanol. The resulting monosaccharides were suspended in 100 ⁇ labeling mix containing 87.1 mg/ml l-phenyl-3-methyl-5 -pyrazolone (PMP), 50% MeOH and 150 mM NaOH. Labeling was performed during 60 minutes at 70°C. The samples were neutralized by the addition of 50 ⁇ 300 mM HC1 and 20 ⁇ 100 mM Tris/HCl pH 7.0. The PMP-labeled monosaccharides were purified by extraction, once with 1 ml di-buthyl ether and three times with 1 ml CHC13.
  • PMP l-phenyl-3-methyl-5 -pyrazolone
  • the PMP derivatized monosaccharides were separated by RP-HPLC (Merck- Hitachi) on a CI 8 Inertsil ODS-3 column (GL Sciences) equipped with a pre-column.
  • RP-HPLC Merck- Hitachi
  • a two- step gradient from 100% buffer A (13% acetonitrile, 87% H20 (0.045% KH2P04, 0.05% triethylamine, pH 7.0) to 50% buffer A / 50% buffer B (21% acetonitrile, 79% H20 (0.045% KH2P04, 0.05% triethylamine, pH 7.0) over 4 minutes to 100% buffer B over 47 minutes was applied at 35°C and a flow rate of 1 ml/min.
  • the injection volume was 50 ⁇ and elution was monitored by online UV-detection at 250 nm.
  • the individual peaks were identified by overlay with chromatograms of the commercially available monosaccharide standards D- glucose (Sigma- Aldrich #G7528), L-rhamnose (Sigma- Aldrich #R3875), N-acetyl-D- glucosamine (Sigma- Aldrich #A8625) and N-acetyl-L-fucosamine (Omicron Biochemicals #FUC-006).
  • E. coli isolates were obtained from the samples following the analysis. E. coli isolates were identified by classical microbiological exclusion and inclusion strategies involving growth on chrome (CPS3) and MacConkey agar. E. coli isolates further were analyzed using an agglutination assay to determine their O antigen serotype. See DebRoy et al. (2011) Animal health research reviews / Conference of Research Workers in Animal Diseases 12, 169-185. Isolates from the same O antigen serogroups were further analyzed to determine the chemical structure of the O chain from each isolate. See Table 1 A. Certain isolated E. coli strains were determined to be antibiotic resistant, including identification of fluoroquino lone-resistant strains and extended- spectrum beta-lactamase (ESBL) producing strains.
  • ESBL beta-lactamase
  • Table 1 A Distribution of the most common UTI-associated E. coli serotypes from a collection of 1841 urine samples collected in Switzerland in 2012. Shown is the serotype distribution of samples from a relevant subpopulation of 671 subjects, and the distribution from all** samples.
  • Serotypes 01, 02, 04, 06, 07, 08, 016, 018, 025, 073, and 075 were isolated from subjects independent of location, time of isolation, symptoms, and target population, suggesting these to be the predominant serotypes of uropathogenic E. coli
  • Table IB Prevalence of most common UTI associated serotypes from selected literature ranging from 1987- 2011 and from retrospectively analysed US data from 2000-2011 (ECRC).
  • Isolates from serotypes described were calculated as percentage on the total number of isolates (Andreu et al, 1997, J Infect Dis 176:464-469; Blanco et al, 1996, Eur J Epidemiol 12: 191-198; Fathollahi et al., 2009, Egyptian Journal of Clinical Infectious Diseases 4:77-81; Johnson et al, 2005, J Clin Microbiol 43:6064-6072; Molina-Lopez et al, 2011, Journal of infection in developing countries 5:840-849; Sandberg et al, 1988, J Clin Microbiol 26: 1471-1476; K. L.
  • O antigen serotypes of E. coli often are composed of subtypes, which are distinct, yet structurally and antigenically similar. To identify unknown/unreported subtypes among the collected clinical isolates, and to identify the most prevalent O antigen subtypes, the chemical structures of the O antigens from the most prevalent serotypes were analyzed in more detail.
  • E. coli E47a is used as a reference strain for 025 serotyping. Further rfb cluster sequence information is available from the genome sequence of a strain causing asymptomatic bacteriuria, E. coli 83972. (see Zdziarskiet al, 2010, PLoS Pathog 6, el001078). Although phenotypic 025 expression has not been confirmed, rfb cluster sequences of E. coli E47a and 83972 are 99.49% identical, strongly suggesting they encode the same O antigen.
  • the O antigen from E. coli strains 83972 and E47a is designated herein as "025A,” because, as described below, a novel E. coli O antigen, designated "025B,” was identified based on analysis of the clinical isolates obtained in the epidemiology study described in Example 1, above.
  • Wzy 0 antigen polymerase Wzy E. coli
  • ADR74237.1 30% wekC Glycosyltransferase Wf F
  • UDP-Glc:FucNAc-GlcNAc-UPP a- 1,3-glucosyltransferase (E. coli)
  • wbuB Glycosyltransferase UDP-L-FucNAc GlcNAc-UPP a- 1,3- N- Acetylfucosaminyltransferase (E. coli) P12b, 026], YP 006169152.1, 73% wbuC Truncated WbuC (E. coli), AAV74548.1, 72%
  • RmlBDAC encode the enzymes required for biosynthesis of dTDP-L-Rhamnose, which is the substrate for the addition of L-Rha branch to the OPS repeat unit.
  • FnlABC encode the enzymes required for biosynthesis of UDP-L-FucNAc, which is the donor substrate for the addition of L-FucNAc to the 025 OPS repeat.
  • wekABC and wbuBC are glycosyltransferases according to homology analysis.
  • Wzx and Wzy are required for flipping of the BRU to the periplasmic space and their polymerization on Und-PP.
  • 025 agglutination positive strains were analyzed by typing PCR for 025 and 025B.
  • PCR was performed using colonies picked from a petri dish as template DNA source and different oligonucleotide primers.
  • 025-specific primers based on amplification of E47a 025 wzy, and described in Li, et al. (2010) J Microbiol Methods 82, 71-77 were used. Also used were the 025B-specific primers described in Blanco, et al.
  • the cluster composition rfb shows clear differences to the composition of the 025 A cluster.
  • the genes in the 5' portion of the cluster are close homo logs of each other (rmlD to wzy; E. coli E47a and 83972). This is not surprising for the rml genes which are homologous in many E. coli strains that synthetize L-rhamnose. Homology of the gene products of 025A and B reaches into the wekC (025 A) gene, before it drops to levels below 25 %> identity, indicating unrelatedness of the protein sequences. See Fig. 3B.
  • the genes identified in the 025B rfb cluster that are not present in the rfb cluster of serotype 025A encode two glycosyltransferases and an O-acetyltransferase. These three genes share the same organization and the encoded proteins have high homology with the wbbJKL genes found and characterized in K-12 strains of E. coli of the 016 rfb cluster genotype. See Fig. 3B. According to the genetic relatedness between the 025B and 016 serotypes, the nomenclature of the 016 rfb genes, wbbJKL, was applied to the homologous genes identified in the 025B rfb cluster.
  • WbbJKL are responsible for acetylation of L-Rhamnose, transfer of a D-Glc residue to L-Rha-D-Glc- UndPP, and the transfer of the L-Rha to D-GlcNAc-UPP, which is formed by wecA from the ECA cluster.
  • WbbJKLo25B synthesize the same structure as WbbJKL 0 i6, i.e. a-D-Glc-l,3-a-L-Rha(2Ac)- 1,3-a-D-GlcNAc.
  • This trisaccharide structure is identical to the unbranched 'core' backbone of 025A with the only exception that the L-Rha(2Ac) replaces the L-FucNAc. The replacement would accordingly be a conservative one, as D-FucNAc and L-RhaOAc are both monosaccharides with a 6-deoxy and 2-acetyl function.
  • LPS Lipopolysaccharide
  • Clinical isolate upecl38 was positive for the 025B by PCR, and exhibits a weaker recognition by the 025 agglutination antiserum than 025 A strains. See Fig. 5.
  • the strain is ESBL, but sensitive to FOS, IPM, and TZP, and resistant to AM, CXM, NOR, and CIP.
  • Another clinical isolate, strain upec436, was negative for 025B by PCR, but positive for the general 025 (025 A) by PCR. upec436 also was found to be strongly reactive with the 025 agglutination antiserum when its LPS was analyzed by Western blotting.
  • the OPS extraction, hydrolysis and 2AB labeling procedure involves acid treatment to remove the Und-PP from the OPS. It was shown that the treatment conditions partially remove O-acetylation, but not N-acetylation. Thus, it is likely that the peak at 60' represents a deacetylated BRU mass that emerged from the chemical hydrolysis of the material in the 50' peak. Taken together, this data indicates that there is an O-acetylation in 025B at the same monosaccharide position as there is N-acetylation in the L-FucNAc of 025A.
  • 025B representative strain upecl38 is structurally and genetically related to the 025 A and 016 OPS (Fig. 3 A and B) from E. coli.
  • 025B differs from 025A in having a repeat unit structure containing an O-acetyl group instead of an N-acetyl group at the second monosaccharide of the repeat unit, which is a L- Rha residue and not a D-FucNAc.
  • These changes were most likely caused by a replacement of the UDP-FucNAc biosynthesis machinery and the D-FucNAc transferase by a DNA stretch encoding two glycosyltransferases and an O-acetyltransferase.
  • These genes are related to the 016 gene cluster, based on homology and functionality analysis. The final structures are different, but similar, explaining the cross-reactivity observed with the 025 agglutination antiserum.
  • the waaL genes from strains upec_436 (025A) and uepc_138 (025B) were deleted as previously described (see Datsenko and Wanner, (2000) Proc Natl Acad Sci U S A 97, 6640-6645) informed by the method for core type determination (see Amor, et al., (2000) Infect Immun 68, 1116-1124).
  • Resulting strains were transformed with expression plasmids for 4S-EPA (pGVXN659) and an oligosaccharyl transferase, PglB (pGVXN939), for 025A production; and with pGVXNl 14 and pGVXN539 (producing 2S-EPA) for production of 025B bioconjugate production.
  • 025B bioconjugates were produced in a 2L shake flask with subsequent affinity purification from periplasmic extracts by IMAC.
  • 025A conjugates were produced by fed-batch fermentation, and purified by a two-step purification procedure starting from clarified whole cell homogenate generated by high pressure homogenization as described in the methods section above. Monosaccharide composition analysis was performed as described above.
  • bioconjugate material was produced.
  • 025 A the purified batch of 025A-EPA from above was used for further characterization experiments.
  • 025B-EPA production a strain with a genomically integrated 025B cluster was constructed: W3110 AwaaL AgtrABS Ar 3 ⁇ 4016::r 3 ⁇ 4(upecl38), with plasmids pGVXN1076 and pGVXN970.
  • This strain was constructed starting from W3110 by the methods of Datsenko and Wanner and a homologous recombination technique for site directed integration of large inserts into bacterial chromosomes (see International Patent Application No. PCT/EP2013/071328).
  • Maltose binding protein was used as a carrier protein, and the carrier protein was linked to 025A or 025B.
  • Table 4 depicts the strains used for protein production. The used strains were identified by PCR for their 025A or B genotype. Expression was performed in TB medium and protein product purified from periplasmic extracts.
  • Structural databases list different subserotype structures for E. coli 01.
  • OIA and 01A1 are structurally identical and believed to be associated with disease, although OIB and C have not been reported to be pathogenic (see Gupta, et al, (1992) J Bacteriol 174, 7963-7970), and represent a minority among Ol isolates.
  • Structures of OIA/OIAI, OIB, and OIC are shown in Fig. 12B.
  • Fig. 12B To analyze the Ol subserotype distribution in the UPEC epidemiology study of Example 1, the O antigen structures of several clinical isolates from the study were analyzed in detail. First, LPS structure of 12 strains determined to be positive for Ol by agglutination assay were analyzed by SDS PAGE. See Fig. 13: 01 silver staining and Western blot.
  • Fig. 14A shows an overlay of the chromatograms obtained from 5 of the 11 clinical isolates.
  • the fingerprinting area of the OPS appears at retention times of 110 to 150 minutes.
  • the profiles indicate that all samples have signals appearing at the same retention times, indicating identical molecule structures. Differences observed were the intensity distribution, i.e. the elution time of the mean maximum signal, and the general signal intensities.
  • the remaining 6 extracts resulted in peaks at the same elution times with differences in intensities. Only sample upecl096 was different with respect to the peak pattern, confirming the structural difference noted above.
  • MS/MS analysis of individual peak contents by MALDI-TOF/TOF analysis was used to identify the sequence of monosaccharides in the 01 samples (see Fig. 14B).
  • Fragmentation ion series obtained after MS/MS were consistent with 1, 2, 3, and 4 repeat units of a HexNAc, three deoxyhexoses, and a branching HexNAc. This data can only be explained by the 01 A subserotype structure.
  • the described peak series represents the 01 OPS attached to UPP in clinical isolates, and every consecutive peak differs to the previous one by one repeat unit.
  • W3110 E. coli strains were engineered to express the OIA OPS.
  • Resulting strains were W3110 ArfbO ⁇ 6 rfbO ⁇ AwaaL, containing the rfb cluster of an 01 positive clinical isolate (GU299791 *, cluster ranging from rmlB-wekO).
  • the OIA OPS expressing host strains were constructed by homologous recombination.
  • the rfb cluster of an OIA clinical isolate was amplified using PCR oligonucleotides annealing in the DNA flanking the rfb cluster.
  • the amplified DNA was then used to replace the endogenous O antigen cluster of the well characterized laboratory strain W3110 by the homologous recombination described in International Patent Application No. PCT/EP2013/071328.
  • Expression plasmids for the carrier proteins pGVXN659 and for PglB (pGVXNl 14, 939, 970, 971) were inserted by transformation and 01 expression was confirmed (see Fig. 15 and Fig. 16).
  • upec032 was engineered to produce bioconjugates.
  • upec032 AwaaL was constructed and transformed with pGVXN939 and pGVXN579 for bioconjugate production using MBP as carrier protein.
  • MBP carrier protein
  • the advantage of using MBP and EPA as carrier proteins is the possibility to raise antisera with both resulting in antisera that are crossreactive towards the polysaccharide component but not the carrier.
  • Such antisera are useful tools for evaluation of preclinical experiments, e.g. as coating agents to develop polysaccharide specific ELISA assays.
  • E. coli 06 serotype is the most frequent ExPEC reported to date (George, D. B., and Manges, A. R. (2010) Epidemiol Infect 138, 1679-1690). Not only the study described in Example 1 , but also data taken from the literature confirms that the 06 serotype is among the top four serotypes in many ExPEC caused manifestations (see Fig. 4).
  • the 06 OPS with a Glc branch (Fig. 17B, top) is the most representative structure among the 06 serotypes isolated from the epidemiology described in Example 1.
  • W3110 E. coli strains were engineered to express the 06 OPS by replacing the W3110 rfb cluster with the rfb cluster from strain CCUG11309. See Table s7 and 13. Resulting strains were W3110 Ar 3 ⁇ 4016::r 3 ⁇ 4CCUGl 1309 AwaaL, containing the rfb cluster of an 06 positive E. coli strain with reported Glc branch in the BRU (see above). The 06Glc OPS expressing host strain were constructed by homologous recombination.
  • the rfb cluster was amplified using PCR oligonucleotides annealing in the DNA flanking the rfb cluster.
  • the amplified DNA was then used to replace the endogenous O antigen cluster of the well characterized laboratory strain W3110 by the homologous recombination described in International Patent Application No. PCT/EP2013/071328.
  • Expression plasmids for the carrier proteins and for PglB were inserted by transformation and expression of the expected OPS on EPA was confirmed by Western blotting.
  • rmlBDAC encode the enzymes required for biosynthesis of dTDP-L-Rhamnose, which is the substrate for the addition of L-Rha to the backbone by glycosyltransferases wekPOR;
  • fdtABC provide dTDP-D-Fuc3NAc for the branching glycosyltransferases
  • wekPOR are predicted glycosyltransferases, and are predicted to form glycosidic linkages of the 02 BRU (three L-Rha and one L-FucNAc).
  • the wekS gene found in the published 02 rfb cluster sequences is a predicted membrane bound sulfatase, and thus most likely is not involved in BRU formation. This would mean that - if one assumes the one enzyme one linkage rule- that one enzyme among the group of wekPOR must be bifuntional to provide the four glycosidic linkages.
  • the O antigen cluster from strain W3110 was replaced by the rfb cluster from the 02 positive strain upecl 16 lacking the wekS DNA. Chromosomal replacement was done by homologous recombination as described in International Patent Application No. PCT/EP2013/071328. The resulting strain was W3110 AwaaL ArfbW3110::r 3 ⁇ 4O2 AwekS. OPS was prepared and analyzed by 2AB labeling and normal phase HPLC and fluorescence detection as done for 01A and 06 OPS (see above) and analyzed by normal phase HPLC (Fig. 21) and compared to the signals from wild type strain CCUG25. Analysis resulted in a series of overlapping peaks between 40 and 140 minutes elution time.
  • Table 6 Overview of the preclinical rat study, including vaccine group numbers, group sizes, vaccines used, and quality indication of the vaccine preparation.
  • bioconjugates selected and produced represent useful vaccine candidate compounds for the induction of O-antigen-specific antibodies.
  • biococonjugates described in the examples above (O-antigen of 025B, 01 A, 02 and 06, respectively conjugated to EPA as a carrier protein) were prepared as monovalent batches (active pharmaceutical ingredients, APIs) or combined in a single preparation as a multivalent vaccine against ExPEC.
  • Various batches were produced: pre-clinical batches, toxicity study batches, and clinical batches.
  • Table 7 indicates host strains used for the production of conjugates.
  • Table 7 Host strains for production of preclinical, toxicology study and clinical batches
  • a MW of 63-67 kDa was determined (theoretical MW of 70.5 kDa, based on amino acids sequence).
  • the measured MW of the EPA moiety was 65-71 kDa.
  • Peak 1 di-glycosylated form.
  • Peak 2 mono -glycosylated form.
  • DSC Differential scanning calorimetry
  • Example 8 Stability of monovalent bulks and tetravalent vaccine preparations
  • S/P sugar to protein ration as determined by anthrone and BCA assays, respectively. Purity: as determined by reverse phase high resolution liquid chromatography (RP- HPLC).
  • Stability of the tetravalent vaccine composition (025B, 01 A, 02 and 06 bioconjugates) was tested during over a 3 month period. The studies included accelerated and stress storage conditions to identify degradation pathways. The resultant data are considered to be relevant for the initial justification of the GMP IMP (investigational medicinal product, the tetravalent ExPEC vaccine composition) shelf life.
  • MW molecular size distribution: two main product peaks (i.e. mono- and di- glycosylated species), as determined by size exclusion high resolution liquid
  • the dose administered i.e. a full human dose equivalent of 4 ⁇ g per O-antigen (16 ⁇ g total O-antigen for the tetravalent vaccine), as administered on days 1 and 14, was considered to be the no-observed- adverse-effect- level (NOAEL) for the tetravalent ExPEC vaccine under the conditions of this study.
  • Example 10 Epidemiology of O-serotypes associated with bacteremia
  • Serotype 025 was most prevalent in the bacteremia population studied; subtyping of fifty-seven isolates by PCR showed that fifty-six (98%) of the 025 serotypes were typeable as 025B. In both target populations (UTI and bacteremia), serotypes 01 , 02, 06, and 025 were identified as the four most prevalent serotypes. Overall, these data confirm that serotype distribution among both urinary tract infection and bacteremia isolates is highly similar and independent of geographical location, time of isolation, and indication. Table 11: Distribution of the most common bacteremia-associated E. coli O-serotypes from a collection of 860 blood isolates collected in the US and EU in the period 2011-2013.
  • OPK opsonophagocytic killing
  • the ability of the antibodies to bind the bacterial cells and activate deposition of the complement and mediate uptake and killing of the bacteria by HL60 cells was expressed as opsonic titer.
  • the opsonic titer or opsonization index (OI) corresponds to the dilution of the sera killing 50% of the bacterial cells.
  • Opsonic indices for pre and post-immune sera are provided. A > than 4-fold increase of OI from pre - to post-immune is considered significant.
  • OPK assays for three serotypes 02, 06Glc and 025B were established. Functionality of antibody responses induced by monovalent vaccines
  • opsonophagocytic killing (OPK) assays which measure in vitro complement- and antibody-dependent phagocytosis and killing of bacteria, e.g., E. coli.
  • OPK opsonophagocytic killing
  • E. coli selected for OPK testing were OC 24453 (serotype 02), OC 24781 (serotype 06Glc) and OC 24176 (serotype 025B).
  • OC 24453 serotype 02
  • OC 24781 serotype 06Glc
  • OC 24176 serotype 025B
  • FIG. 29A a robust functional immune response to 02-EPA
  • FIG. 29B a robust functional immune response to 02-EPA
  • 06Glc-EPA FIG. 29B
  • 025B-EPA FIG. 29C
  • Table 12 shows the total OI titers for the O-antigens 02, 06Glc and 025B from animals immunized with the tetravalent vaccine with either 0.4 or 4 ⁇ g per O-antigen.
  • the titers were determined in two separate experiments.
  • the 0.4 ⁇ g dose induced significant OIs in all animals for the 02 and 06Glc serotypes.
  • For 025B 3/8 animals showed a significant increase in 01 following immunization with the 0.4 ⁇ g dose.
  • the 4 ⁇ g dose induced lower 01 increases for 02 in all animals.
  • 3/8 animals showed 01 increases when the sera from the 4 ⁇ g dose group were tested on 025B E. coli.
  • the data confirm that a tetravalent vaccine is able to elicit O-antigen-specific opsonic antibodies against 02, 06Glc and 025B.
  • Ill Table 12 OIs against E. coli 02, 06 and 025. OIs for individual pre -vaccination and post 3 vaccination sera from two separate experiments are shown for all animals.
  • E. coli selected for OPK testing were OC 24453 (serotype 02), OC 24781 (serotype 06Glc) and OC 24176 (serotype 025B). Robust functional immune response to 02-EPA, 06Glc-EPA and 025B-EPA was observed.
  • Example 12 Evaluation of a Candidate Vaccine against Uropathogenic Escherichia Coli in Women with a Clinical History of Recurrent Urinary Tract Infection (RUTI)
  • E. coli bioconjugate vaccine is used in a Phase I clinical study.
  • the vaccine comprises four bioconjugates in a saline buffer solution.
  • the four bioconjugates are: (i) E. coli 01 A conjugated to EPA carrier protein, (ii) E. coli 02 conjugated to EPA carrier protein, (iii) E. coli 06Glc conjugated to EPA carrier protein, and (iv) E. coli 025B conjugated to EPA carrier protein.
  • the study population includes 194 healthy females, aged > 18 to 70 years old, with a history of recurrent urinary tract infection (RUTI), defined as > 3 independent episodes in the previous 12 months or > 2 episodes in the last 6 months.
  • RUTI recurrent urinary tract infection
  • At least one of the urinary tract infection (UTI) episodes was caused by E. coli (as single pathogen or part of polymicrobial infection) and the cause was culture-confirmed and documented.
  • UTI is defined by the presence of at least one specified UTI symptom (dysuria, urgency, frequency, flank pain, bladder tenderness, suprapubic pain, fever, nausea, vomiting) along with a bacterial count (CFU) of >10 J CFU/ml uropatho gen in mid-stream urine.
  • CFU bacterial count
  • the study includes two arms: (i) candidate vaccine and (ii) placebo.
  • the study is a staggered, randomized, single blind, placebo-controlled multi-center study in healthy women with history of RUTI.
  • the estimated enrollment period for the study is 4 months, with a follow-up duration period of nine months for each subject.
  • the objective of the study is to assess the safety, immunogenicity, and efficacy of the E. coli bioconjugate vaccine.
  • Subjects are followed for 9 months after injection, and only injected subjects are followed throughout the study period. Subjects attend a total of 5 scheduled visits: screening (first visit), day 1 (second visit), day 7, day 30, and day 270. Subjects receive 4 follow-up phone calls, on day 2, day 90, day 150, and and day 210.
  • Any unscheduled visits due to occurrence of UTI include standard of care with harmonized treatment options.
  • Urine and blood are collected for diagnostic and serotyping purposes.
  • Unsolicited adverse events (AE) and severe adverse events (SAEs) are recorded along the study duration whereas solicited AE are recorded for 7 days after injection.
  • each subject will receive one intramuscular injection of 0.5 ml of solution (vaccine candidate or placebo) in the deltoid muscle.
  • the reduced dose of the candidate vaccine will contain 1 ⁇ g of each polysaccharide (total 4 ⁇ g polysaccharide).
  • the target dose of the candidate vaccine will contain 4 ⁇ g of each polysaccharide (total 16 ⁇ g polysaccharide).
  • the primary objective is to assess the occurrence, intensity, relationship, and duration of solicited and unsolicited adverse events (AE) and serious adverse events (SAE) post-injection of candidate vaccine ccompared to the placebo group throughout the study period.
  • the secondary objectives are (i) to compare the change in hematological and biochemical safety endpoints prior to injection (at the initial screening and day 1) and post injection (at day 7 and day 30) of candidate vaccine compared to the placebo group; (ii) evaluate the immune-response of candidate vaccine between baseline (day 1) and post injection (day 30 and day 270); (iii) compare the number of symptomatic urinary tract infection (UTI) episodes caused by the E.
  • UTI symptomatic urinary tract infection
  • the exploratory objectives are (i) to compare the rate of UTI occurrence caused by any E.coli serotype in the vaccine group compared to the placebo group throughout the study period; and (ii) to compare the rate of UTI occurrence caused by any pathogens in the vaccine group compared to the placebo group throughout the study period.
  • Inclusion criteria for the study are as follows: (i) female subjects with a history of recurrent UTI, which is defined as: > 3 UTI independent episodes in the previous 12 months or > 2 UTI episodes in the last 6 months; at least one UTI during the last 5 years was caused by E.
  • Exclusion criteria for the study are as follows: (i) history of more than 10 recurrent UTIs in the year before the screening visit; (ii) use of any short-term urinary catheter within 7 days prior to screening; (iii) use of any permanent catheter within 30 days prior to screening; (iv) history of any unresolved urinary tract diseases/abnormalities; (v) evidence of impaired immune function; (vi) significant cardiovascular, liver, renal diseases and/or insufficiency; (vii) uncontrolled diabetes mellitus; (viii) significant abnormalities in screening results for
  • corticosteroids >0.5 mg/kg BW/day); (xiii) use of UTI-related vaginal estrogen treatment newly started less than 6 months before injection and continuing during the study or planned start during the active study period; (xiv) use of any antibiotic therapy within 1 week preceding injection; (xv) planned use of post-coital antibiotics for UTI prevention during study period; (xvi) any vaccination planned within 30 days before and 30 days after injection; (xvii) participation in other clinical trials in the 60 days preceding enrolment and for the duration of the study; (xviii) previous treatment with immunoglobulins or blood products in the 3 months preceding the injection; (xix) known hypersensitivity to any component of the vaccine; (xx) presence of a significant medical or psychiatric condition that in the opinion of the investigator precludes participation in the study; (xxi) acute illness at the time of injection; (xxii) women of child bearing potential who either have a positive pregnancy test or refuse to use an effective contraception; (xxiii) women who are
  • Descriptive statistics (n, mean, standard deviation, median and ranges for continuous variables, frequencies and percentages for categorical variables) are provided by treatment group and/or visit, where applicable. All data are listed by subject, treatment group and, where applicable, visit. All subjects from Group B receiving placebo are combined to form the placebo treatment group.
  • OPK assays which measure in vitro complement- and antibody-dependent phagocytosis and killing of E. coli bacteria, were used to assess the the functional antibody response of women participating in the clinical study.
  • E. coli was pre-opsonized with dilutions of serum from the vaccinated women, incubated with complement and phagocytes (differentiated HL60 cells), and the remaining colony forming units (CFUs) was determined. Subsequently, the maximum percent killing and Opsonization Indices (OI: serum dilution killing of 50% of E. coli) were calculated.
  • E. coli selected for OPK testing were OC 24452 (serotype 01 A), OC 24453 (serotype 02), OC 24454 (serotype 06Glc), and OC 24176 (serotype 025B).
  • Tables 13 and 14, below, provide details of certain strains and plasmids, respectively, used in the foregoing examples.

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Abstract

La présente invention concerne un nouveau polysaccharide O d'E. coli, O25B. L'invention concerne également des cellules hôtes procaryotes comprenant des enzymes (par exemple, des glycosyltransférases) utilisées dans la production d'O25B. Les cellules hôtes de l'invention produisent des bioconjugués d'O25B, lesdits bioconjugués comprenant O25B lié à une protéine porteuse. L'invention concerne en outre des compositions, par exemple, des compositions pharmaceutiques, comprenant O25B et/ou des bioconjugués comprenant O25B. Ces compositions peuvent être utilisées comme vaccins contre une infection par ExPEC, et peuvent en outre comprendre un ou plusieurs bioconjugués supplémentaires.
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